Van Andel Research Institute
Scientific Report 2005
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333 Bostwick Avenue, N.E., Grand Rapids, MI 49503 Phone (616) 234-5000; Fax (616) 234-5001; Web site: www.vai.org
Cover photograph of the Van Andel Institute building, Grand Rapids, Michigan
Van Andel Research Institute Scientific Report 2005
Title page photo: Podosomes in transformed cells This image shows podosomes in NIH3T3 mouse fibroblasts transformed with activated Src tyrosine kinase. Podosomes are the rounded structures at the tips of many of the cell extensions. They are rich in filamentous actin, which has been stained green with a conjugated phalloidin dye. The cells are co-stained with an antibody that recognizes the adhesion protein CrkL (red; or where co-localized with actin and its phalloidin stain, yellow). The protein CrkL is involved in integrin-induced cell adhesion and migration. The study of these proteins (Src, F-actin, CrkL, integrins) in these and other tumorigenic cell types may shed light on the mechanisms of podosome-mediated cancer cell metastasis and invasion. (Eduardo F. Azucena, Jr., Darren Seals, James Resau, and Sara Courtneidge)
Published June 2005 Š2005 by the Van Andel Institute All rights reserved
Van Andel Institute 333 Bostwick Avenue, N.E. Grand Rapids, Michigan 49503, U.S.A.
Contents Director’s Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Laboratory Reports Cell Structure and Signal Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Arthur S. Alberts, Ph.D. Antibody Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Brian Cao, M.D. Mass Spectrometry and Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Gregory S. Cavey, B.S. Signal Regulation and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Sara A. Courtneidge, Ph.D. Cancer and Developmental Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Nicholas S. Duesbery, Ph.D. Vivarium and Transgenics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Bryn Eagleson, A.A., RLATG Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Kyle Furge, Ph.D. Cancer Immunodiagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Brian B. Haab, Ph.D. Molecular Medicine and Virology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Sheri L. Holmen, Ph.D. Integrin Signaling and Tumorigenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Cindy K. Miranti, Ph.D. Analytical, Cellular, and Molecular Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 and Microarray Technology and Molecular Diagnostics James H. Resau, Ph.D. Germline Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Pamela J. Swiatek, Ph.D., M.B.A. Cancer Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Bin T. Teh, M.D., Ph.D.
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Molecular Oncology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 George F. Vande Woude, Ph.D. Tumor Metastasis and Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Craig P. Webb, Ph.D. Chromosome Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Michael Weinreich, Ph.D. Cell Signaling and Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Bart O. Williams, Ph.D. Structural Sciences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 H. Eric Xu, Ph.D. Mammalian Developmental Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Nian Zhang, Ph.D. Daniel Nathans Memorial Award . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Postdoctoral Fellowship Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Student Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Han-Mo Koo Memorial Seminar Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Recent VARI Photos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
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Director’s Introduction
Director’s Introduction Institute, the Research Institute is developing a graduate school with a program leading to the Ph.D. degree in cellular and molecular genetics, with an emphasis on translation. We expect the graduate program to contribute to the vitality and creativity of our successful research programs and to also address the nation’s need for expertise in the life sciences and biotechnology. We have received a charter from the state of Michigan and have appointed a Board of Directors for the school. Our goal is to have our first students on board in September 2006.
We are now in our fifth year since the opening of the Van Andel Institute. The Institute was created because of the generosity of Jay Van Andel and his wife Betty, both of whom George F. Vande Woude passed away in 2004. While deeply saddened by their loss, we continue our endeavor to fulfill their vision of a center for research and education excellence in the heart of Grand Rapids.
Personnel A special occasion occurred this past year with our first promotion review. We are very proud to announce the promotion of Bin Tean Teh to Distinguished Scientific Investigator, our highest appointment level. Bin has made major contributions to the understanding of kidney, nasopharyngeal, and endocrine cancer. In recognition of his efforts, Bin was recently appointed to the Medical Advisory Board of the Kidney Cancer Association. We are also proud to announce the appointment of Rick Hay as a Senior Scientific Investigator. Rick will establish the Laboratory of Animal Imaging. Our congratulations to both scientists!
I know Jay was able to see the beginning of what a great contribution he made to society and the betterment of human health. Sharing his and Betty’s vision, we have begun to expand our research beyond cancer biology into the field of neurological disorders. David and Carol Van Andel, in Jay’s honor, have created an endowed chair dedicated to Parkinson disease (PD) research. The search for a leading scientist to take this chair is now underway. Already, Jim Resau and Bin Teh have initiated a collaboration with Australian scientists Alan Mackay-Sim and Peter Silburn at Queensland University in Australia to better understand Parkinson disease and how it develops. Alan is a developmental biologist who has initiated some exciting discoveries using adult olfactory stem cells. Peter is a neurologist who studies and treats PD patients. Together with these new collaborators, we will increase our understanding of the function, growth, and death of human nerve cells as models for PD. In addition, these approaches will be used in the study of Alzheimer disease pathophysiology. Our scientists have also partnered with the St. Mary’s Hauenstein Parkinson Center—named in honor of lead donor (and VAI trustee) Ralph Hauenstein—to determine the interactions of the environment and genes associated with PD. We will learn how these genes correlate with disease progression and drug response.
Sara Courtneidge has taken a position at the Burnham Institute and is relocating her laboratory in early summer 2005. Sara and I have been friends and colleagues for over 20 years. I was delighted when she joined VARI and I am grateful for all her contributions to the Institute, especially her efforts in helping to establish the graduate program with MSU and the VARI postdoctoral advisory committee. The Burnham Institute is very fortunate to have recruited her, where she will be reunited with her many West Coast friends. I am sorry to see her leave, but I wish her great success and look forward to our continued scientific interactions. Publications and Competitive Funding As of April 1, 2005, there have been 190 peer-reviewed articles published by VARI investigators. In December 2004, a VARI article was featured on the cover of the Journal of Bone
We are pleased to announce that, in collaboration with the Van Andel Education
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of Veterans Affairs Healthcare System in Ann Arbor, ApoLife, Inc., and the National Cancer Institute. A second grant was awarded to Bin Teh for the development of the “RenoChip,” a diagnostic and prognostic tool for use against kidney cancer. In addition, the Department of Defense awarded Eric Xu a grant for a three-year study of the structure and function of the androgen receptor in prostate cancer. Eric’s lab aims to make progress in understanding the androgen dependence (or independence) of prostate cancer.
and Mineral Research; in January 2005, we had a featured article in Molecular Cell; and in February, a VARI article in Cancer Cell was the source of the issue’s cover photo. Our investigators have also demonstrated their competitive research abilities in terms of receiving grants for funding of their work. In fiscal year 2004, extramural funding for VARI dramatically increased over that in 2003. Seventeen of our scientists and six of our postdoctoral fellows received funding from 42 grants. Our new major awards have come from a variety of sources. The National Institutes of Health (NIH) National Cancer Institute (NCI) made two awards to Nick Duesbery. The first was an R01 grant for studying MEK signaling in sarcoma growth and vascularization. The second grant, an R21, was for investigating the antitumor effects of an anthrax toxin moiety (which we have termed tumor lethal factor, or “TLF”) on Kaposi sarcoma. Nick’s lab has been developing TLF as a potential cancer therapeutic. Brian Haab was awarded an R21 NCI grant for a two-year project entitled “Longitudinal CancerSpecific Serum Protein Signatures.” This project seeks to develop protein microarray methods for detecting and diagnosing prostate cancer by examining changes over time in several cancerrelated proteins in serum. And, Art Alberts received an R21 award from the NIH to exploit a discovery in his lab with a long-term goal of developing novel anti-cancer therapies.
Funding from other sources in the past year has included Brian Haab’s grant from DHHS/ NCI via the University of Michigan for a project entitled “Accelerated Cancer Biomarker Discovery.” This project is being undertaken by a consortium of laboratories and focuses on the development and application of new proteomics technologies for cancer biomarker discovery. Bin Teh has received a grant from the Schregardus Family Foundation for a project on renal cell carcinoma (RCC) in which his lab will be studying the prognostic value of genes for improving the clinical management of RCC patients. Bin is also the recipient of a grant from the Gerber Foundation for gene expression profiling in newborns with congenital chromosomal abnormalities. We are also proud that three of our postdoctoral fellows have received awards. Carrie Graveel (Vande Woude lab) and Kate Eisenmann (Alberts lab) have received National Research Service Awards from the NIH, while Jennifer Bromberg-White (Webb lab) received a fellowship award from the Multiple Myeloma Research Foundation.
The American Cancer Society awarded two Research Scholar Grants to our researchers. One grant, to Art Alberts, is for a four-year project to study Diaphanous-related formins in myelodysplasia. Art has been studying the role of formins in cancer. A second American Cancer Society grant was awarded to Michael Weinreich. Michael is identifying small-molecule inhibitors of Cdc7 kinase for study of its regulation in DNA replication, with a long-term goal of identifying novel targets for cancer diagnosis and therapy.
Looking to the Future We now look to expanding not only our research goals but the Institute itself. On May 17th we celebrated our fifth anniversary, and our CEO, David Van Andel, announced that in 2006 we will begin the second construction phase of our Institute. The new building, a model of which is displayed in the Cook-Hauenstein Hall, will join and mirror our current exceptional facility, but it will provide two-and-a-half times the existing laboratory space, or an additional 150,000 square feet.
Two major grants have also been received from the Michigan Technology Tri-Corridor (MTTC). One award went to Rick Hay for the development of novel agents for nuclear imaging and therapy of Met-expressing human tumors. The project is a collaboration among scientists at VARI, Michigan State University, the Department 4
clinical-grade biological products that can be tested in patients. Finally, there is the strong possibility of Michigan State University’s College of Human Medicine relocating to Grand Rapids, which would certainly be a major event in the development of the biomedical community here.
As we plan and begin our expansion, we are part of the unprecedented growth in the health industry that Grand Rapids is experiencing. The area of Grand Rapids in which the Institute is situated has been appropriately renamed “Medical Hill.” Our neighbor across Bostwick Avenue, Spectrum Health, has celebrated the opening of the new Fred and Lena Meijer Heart Center. Furthermore, the hospital will soon break ground for the construction of a new cancer center as well as a new pediatric hospital. In another project, our own Rick Hay serves as the chairman of a combined VARI and Grand Valley State University group that is planning a good manufacturing practices (GMP) facility. This facility is being established with state and federal funding, and it will produce small quantities of
Overall, with the new hospital facilities at Spectrum Health and St. Mary’s, Grand Valley State University’s strength in health sciences, and our own research program and future expansion, we will witness in the next decade exciting and dramatic developments in biomedical research, scientific discovery, and health care delivery taking place in Grand Rapids. We look forward to these many exciting changes and to the formation of a center of excellence in biomedical disciplines in western Michigan.
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Van Andel Research Institute Laboratory Reports
2005 VARI Scientific Retreat
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Laboratory of Cell Structure and Signal Integration Arthur S. Alberts, Ph.D. Dr. Alberts received his Ph.D. in physiology and pharmacology at the University of California, San Diego, in 1993, where he studied with James Feramisco. From 1994 to 1997, he served as a postdoctoral fellow in Richard Treisman’s laboratory at the Imperial Cancer Research Fund in London, England. From 1997 through 1999, he was an Assistant Research Biochemist in the laboratory of Frank McCormick at the Cancer Research Institute, University of California, San Francisco. Dr. Alberts joined VARI as a Scientific Investigator in January 2000. Laboratory Members
Staff Art Alberts, Ph.D. Jun Peng, M.D. Yunju Chen, Ph.D. Kathryn Eisenmann, Ph.D. Holly Holman, Ph.D. Susan Kitchen, B.S.
Students Aaron DeWard, B.S. Yaojian Liu, B.S. Katharine Collins
Visiting Scientists Stephen Matheson, Ph.D. Brad Wallar, Ph.D.
Research Interests Rho guanine nucleotide exchange factors (Rho GEFs), one of which is the neuroepithelioma transforming gene 1 (NET1). Recently it was demonstrated that wild-type NET1 is localized in the nucleus and that truncation of the amino terminus results in relocalization of a fraction of the NET1 to the cytoplasm. This is at least partially due to the elimination of two putative nuclear localization signals within the amino terminus. Thus, NET1 activity is regulated at least in part through subcellular localization.
T
he actin cytoskeleton is a dynamic, tightly regulated protein network that plays a crucial role in mediating diverse cellular processes including cell division, migration, endocytosis, vesicle trafficking, and cell shape. The research focus of the lab is the genetics and molecular biology of the Rho family of small GTPases and their effectors, which together control multiple aspects of cytoskeletal dynamics. The guiding hypothesis of the laboratory is that cytoskeletal dynamics defines the what, where, and how of signal transduction pathways, control responses to growth factors, and other extracellular cues, and that defects in these tightly controlled dynamics can contribute to cancer pathophysiology. Support for this hypothesis is observed in human cancers that carry mutations in genes encoding regulators of Rho GTPase activity. Ultimately, our goal is to exploit our understanding of the mechanics of GTPase-effector relationships in order to develop anti-cancer therapeutics.
Rho family members can modulate the activity of other Rho proteins. The protein kinase PAK1 down-regulates the activity of the RhoAspecific GEF NET1. Specifically, PAK1 phosphorylates NET1 on three sites in vitro: serines 152, 153, and 538. Replacement of serines 152 and 153 with glutamate residues reduces the activity of NET1 as an exchange factor in vitro, as well as its ability to stimulate actin stress fiber formation in cells. Using a phospho-specific antibody, PAK1 can be shown to phosphorylate NET1 on serine 152 in cells, and Rac1, which activates PAK1, stimulates serine 152 phosphorylation in a PAK1-dependent manner. Furthermore, coexpression of constitutively active PAK1 inhibits NET1 stimulation of actin polymerization only when serines 152 and 153 are present. These observations provide a novel mechanism for the control of RhoA activity.
PAK1 negatively regulates the activity of the Rho exchange factor NET1 Rho GTPases act as molecular switches in cells, alternating between on and off states while bound to GTP and GDP nucleotides, respectively. The activated, GTP-bound proteins preferentially interact with numerous autoregulated downstream effector proteins. Rho GTPases are activated by
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Signal transduction and spatially controlled assembly of F-actin networks
domain (DAD) in the carboxy terminus. The GBDs can interact with their internal DAD partners in vitro, leading to the autoregulation model depicted in Fig. 1B. The model shows that while the formin proteins dimerize through their FH2 domains, it is the GBD-DAD interaction that is the linchpin of autoregulation. GTP-bound Rho can interact with the GBD and interrupt the autoinhibited conformation, leading to nucleation and elongation of nonbranched actin filaments.
One well-characterized actin nucleator, the Arp2/3 complex, induces the formation of branched actin filaments. Arp2/3 works by complexing with G-actin or by binding to the side of preexisting filaments. Its nucleation and filament-binding activity is tightly regulated by interactions with nucleation-promoting factors (NPFs), the most prominent being the WASp/Scar family. WASp is an autoregulated molecular switch controlled by yet other switch-like proteins such as Cdc42, a Rho family small GTPase. Thus, NPFs integrate signals controlling growth factor–stimulated actin nucleation and branching. Cdc42-activated WASp induction of Arp2/3 activity is shown schematically in Fig. 1A.
It has been unclear whether GTPase binding simply activates the mDia proteins’ ability to nucleate actin, or provides other signals that direct subcellular targeting and recruitment of mDia-associated proteins. Another question is, do mDia proteins activated in specific cellular contexts (i.e., on vesicles or at sites of adhesion) associate with or work in parallel with other modifiers of actin polymerization to generate site-specific F-actin networks? We are studying such questions using FRET technology.
The mammalian Diaphanous-related formins Formins are a highly conserved family of proteins implicated in a diverse array of cellular functions including the cytoskeletal remodeling events necessary for cytokinesis, bud formation in yeast, establishment of cell/organelle polarity, and endocytosis. Formins have the ability to stabilize microtubules, which (like F-actin) are assembled by tightly controlled cycles of polymerization and depolymerization.
Site-specific interactions between Rho GTPases and mDia proteins Fluorescence resonance energy transfer (FRET) is a powerful technique that allows us to assay protein-protein interactions in cells by using two fluorophores, in this case, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). When fused to the GTPase and excited at the appropriate wavelength, CFP acts as a fluorescent donor which then excites YFP, which is fused to a Drf protein (Fig. 1B). FRET
The mammalian Diaphanous-related formin (mDia) proteins are a subfamily of formins that share a loosely conserved Rho GTPase-binding domain (GBD) in the amino terminus and a highly conserved Diaphanous-autoregulatory
Figure 1. Drfs are actin nucleators whose activity is regulated through interactions with small GTPases. A) A model for formin (mDia1-3) and WASp collaborating in cells with activated Cdc42, in which Cdc42 interacts with mDia2 in cells at specific sites associated with membrane protrusions. In this model, activated WASp nucleates branched filaments from the side of mDia2 nucleated “mother” filaments; alternatively, mDia2 binds to and processively elongates filaments after nucleation by Arp2/3. B) GTP-bound Rho GTPase binding disrupts intramolecular interactions between the GBD and DAD of a DRF. If the GTPase and GBD are linked to fluorophores (ECFP and EYFP), the proximity of the two can be determined by FRET.
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Figure 2. RhoB interacts with mDia2 on vesicles. CFP-fused activated RhoB-G14V and YFP-fused mDia2 were co-injected into cells and FRET was assessed 4 h later. A FRET signal is observed between activated RhoB and mDia2 upon endosomes. These data indicate that individual GTPase-formin pairs would appear to be functionally distinct, promoting the formation of different actin structures at different sites. Since both RhoB and Cdc42 have been implicated in endocytosis and vesicle trafficking, it is possible that both GTPases use mDia2 sequentially or in parallel as effectors to transport cargo within cells.
occurs only when the donor/acceptor pair is in close proximity (less than 30 Å) . Fusion proteins are expressed following microinjection of their expression plasmids into cells, which are then fixed 4 h later. We have shown that YFP-mDia2 is expressed with CFPCdc42, primarily at the leading cell edge (or cortex) and at the microtubule-organizing center. In other experiments, we have shown that this interaction depends upon the integrity of the CRIB motif within the mDia2 GBD. CRIB motifs are necessary for binding to the GTPase. Our observations indicate that one particular GTPase-formin pair, Cdc42 and mDia2, may have a role in remodeling actin at the cell edge. What this pair contributes to actin or microtubule dynamics at the MTOC, however, remains an open question. We speculate that the pair may participate in microtubule regulation at the minus (–) end of the tubules, which (like actin) are assembled and disassembled in a polarized fashion. Other GTPase-formin pairs may be working at the plus end to direct them to focal adhesions or other sites. From collaborative efforts with the Gundersen lab, it has been shown that mDia1 and mDia2 can complex with the MT(+)-end binding proteins APC and EB1.
Previously, we found that a peptide derived from the DAD region of mDia proteins, when expressed in cells, stabilized both the actin and microtubule cytoskeletons. Because the mechanism of DAD action is unique—it binds to cellular formins and disrupts their normal autoregulatory mechanism—DAD represents a novel class of anti-tumor drugs.
In contrast to the speculative role of Cdc42 and mDia2, RhoB is known to have a role in endocytic or vesicular trafficking, and it interacts with mDia2 on endosomes (Fig. 2). This result is consistent with our discovery of both mDia1 and mDia2 on endosomes. The expression of either activated RhoB or deregulated versions of mDia1, mDia2, and mDia3 blocks the movement of vesicles and increases their number. One interpretation is that expression of RhoB or deregulated mDia1–3 triggers an inappropriate transition from fast microtubule-dependent transport to actin-dependent transport.
Because DAD is unable to enter cells as a drug, we have begun searching for functional analogs of DAD that could have similar properties under a drug-development program funded by the National Cancer Institute. We hypothesize that by deregulating specific mDia molecules in tumor cells, we can arrest dynamic remodeling of the cytoskeleton, a process required for cell motility and cytokinesis. We will characterize the structural and functional requirements for DADinduced cell death and develop a high-throughput screen for the identification of novel molecules that can deregulate mDia proteins and kill tumor cells. Our objectives are to initiate a drug discovery program by validating mDia proteins as molecular targets in cancer and to determine the physical requirements for DAD interactions with the mDia GBD.
Formins as anti-cancer drug targets A dynamic cytoskeleton is required for tumor cell growth. Drugs that stabilize the cytoskeleton are emerging as effective anti-cancer therapeutics. For example, Taxol binds directly to the components that comprise the microtubule cytoskeleton and blocks their dynamics. A cyclopeptide derived from a sea sponge, jasplakinolide, has similar effects on actin. 11
External Collaborators Philippe Chavrier, Institut Curie, Paris, France Jeff Frost, University of Houston, Texas Gregg Gundersen, Columbia University, New York George Prendergast, Lankenau Institute, Wynnewood, Pennsylvania Kathy Siminovitch, University of Toronto, Canada
Recent Publications Alberts, A.S., H. Qin, H.S. Carr, and J.A. Frost. In press. PAK1 negatively regulates the activity of the Rho exchange factor NET1. Journal of Biological Chemistry. Eisenmann, K.M., J. Peng, B.J. Wallar, and A.S. Alberts. In press. Rho GTPase-formin pairs in cytoskeletal remodeling. In Signaling Networks in Cell Shape and Motility, London, U.K.: Novartis Foundation. Wen, Ying, Christina H. Eng, Jan Schmoranzer, Noemi Cabrera-Poch, Edward J.S. Morris, Michael Chen, Bradley J. Wallar, Arthur S. Alberts, and Gregg G. Gundersen. 2004. EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration. Nature Cell Biology 6(9): 820–830.
Left to right: Liu, Holman, DeWard, Chen, Peng, Collins, Alberts, Kitchen, Eisenmann
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Laboratory of Antibody Technology Brian Cao, M.D. Dr. Cao obtained his M.D. from Beijing Medical University, People’s Republic of China, in 1986. On receiving a CDC fellowship award, he was a visiting scientist at the National Center for Infectious Diseases, Centers for Disease Control and Prevention (1991–1994). He next served as a postdoctoral fellow at Harvard (1994–1995) and at Yale (1995–1996). From 1996 to 1999, Dr. Cao was a Scientist Associate in charge of the Monoclonal Antibody Production Laboratory at the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute–Frederick Cancer Research and Development Center, Maryland. Dr. Cao joined VARI as a Special Program Investigator in June 1999. Laboratory Members
Staff Ping Zhao, M.S. Tessa Grabinski, B.S.
Visiting Scientist Mei Guo, M.S.
Students Yong-jun Jiao Xin Wang Jin Zhu
Research Interests designated Met3 and Met5, to study mouse xenograft and orthotopic models of localized and metastatic prostate cancer via clinical nuclear imaging. Moreover, we will soon be testing these two radiolabeled mAbs on dog spontaneous prostate cancer and bone metastasis models.
H
epatocyte growth factor/scatter factor (HGF/SF) is a multifunctional heterodimeric protein produced by mesenchymal cells and is an effector of cells expressing the tyrosine kinase receptor Met. Met, the protein product of the c-met protooncogene, is from the same family as epidermal growth factor (EGF) receptors. The activation of Met by HGF/SF affects downstream signaling pathways (including other protein kinases) responsible for cellular differentiation, motility, proliferation, organogenesis, angiogenesis, and apoptosis. Aberrant expression of the Met-HGF/SF receptor-ligand complex—resulting either from mutations in the complex or in conjunction with mutations in other oncogenes—is associated with an invasive/metastatic phenotype in most solid human tumors. Met-HGF/SF and downstream kinases are therefore attractive targets for new anti-cancer agents for clinical diagnosis, prognosis, and treatment.
In collaboration with the Nanjing Medical University of China, we have initiated a project to construct a phage-display antibody fragment library. This technique involves the construction and use of animal/human, immunized/naïve Fab and scFv antibody gene repertoires by phage display. The ability to co-select antibodies and their genes allows the isolation of high-affinity, antigen-specific mAbs derived from either immunized animals or non-immunized humans. A number of procedures for selecting such antibodies from recombinant libraries have been described, and some useful antibodies have been produced with this approach. Over the past two years, we have closely followed the development of this technology for producing novel recombinant antibody-like molecules. We have constructed a human naïve Fab library with the diversity of 2 × 109 and have screened out some mAb fragments against tumor marker proteins. In particular, we have selected from this library and characterized one specific anti-Met Fab fragment, designated as Fab-Met-1, using a subtractive whole-cell panning approach (Figs. 1 and 2).
The aberrant expression of the Met receptor kinase by two-thirds of localized prostate cancers, and apparently by all osseous metastases, suggests that Met provides a strong mechanism of selection for metastatic development. We have generated and characterized several anti-Met murine monoclonal antibodies (mAbs) that have high affinity for and specifically recognize Met extracellular domains in their native conformation. In collaborative studies, we are using two of these radiolabeled anti-Met mAbs,
We have also established the technology of a phage-display peptide library for mAb epitope 13
mapping. A random peptide library is constructed by genetically fusing oligonucleotides to the coding sequence of a coat protein of bacteriophage, resulting in display of the fused polypeptide on the surface of the virion. Phage display has been used to create a physical linkage between a vast library of random peptide sequences and the DNA encoding each sequence, allowing rapid identification of peptide ligands for a variety of target molecules such as antibodies. A library of phage is exposed to a plate coated with mAb. Unbound phages are washed away, and specifically bound phages are eluted by lowering the pH. The eluted pool of phage is amplified, and the process is repeated for two more rounds. Individual clones are isolated, screened by ELISA, and sequenced. We have successfully epitope-mapped a variety of important mAbs including anti-HGF/SF, anti-Met, and anti-anthrax lethal factor. We are now exploring the use of this technology on protein-protein interactions; one example is the mapping of the HGF/SF-Met binding site in an in vitro system, and several interesting peptides have been selected from the library as being potential Met antagonists.
Figure 1. A) SDS–PAGE of FabMet-1 fragment purified by affinity chromatography. Lane 1, standard molecular weight markers; lane 2, purified Fab fragment under reducing conditions. The concentration of the running gel was 12%. B) Immunoprecipitation analysis of Fab-Met-1. Met from cell extracts was immunoprecipitated with purified Fab-Met-1 and detected by western blot analysis. Lane 1 is S114 cell lysate immunoprecipitated with C-28 (rabbit anti-human Met polyclonal antibody). Lanes 2–5 are cell lysates immunoprecipitated with Fab-Met-1: lane 2, S-114 (Met+); lane 3, MKN45 (Met+); lane 4, M14 cell (Met–); and lane 5, NIH3T3 (Met–).
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B
histochemistry, immunofluorescence staining, FACS, or in vitro bioassays; production of bulk quantities of mAbs using high-density cell culture; purification of mAbs using FPLC affinity columns; generation of bi-specific mAbs by secondary fusion; conjugation of mAbs to detection enzymes (biotin/streptavidin, fluorescence reporters, etc.); and the development of detection methods/kits such as sandwich ELISA. Over the past few years, this facility has generated more than 200 different mAbs, 10 of which have been licensed to commercial companies. We have also contracted services to local biotechnology companies to generate, characterize, produce, and purify mAbs for their research/diagnostic kit development.
Functioning as an antibody production core facility at the Van Andel Research Institute, this lab has extensive capabilities in the generation, characterization, scaled-up production, and purification of mAbs using comprehensive cutting-edge technologies. The technologies and services available in the core include animal immunization and antigen preparation; peptide design; DNA immunization (Gene-gun technology); immunization of a wide range of antibody-producing models (including mice, rats, rabbits, human cells, and transgenic or knock-out mice); and in vitro immunization. Other services we provide include the generation of hybridomas from spleen cells of immunized mice, rats, and rabbits; hybridoma expansion and subcloning; cryopreservation of hybridomas secreting mAbs; monoclonal antibody isotyping; ELSIA screening of hybridoma supernatants; monoclonal antibody characterization by immunoprecipitation, Western blot, immuno-
S114
M14
Figure 2. The binding affinity of a selected Fab fragment (Fab-Met-1) was tested by FACS analysis. Two cell lines, S114 (Met+) and M14 (Met–), were incubated with purified Fab-Met-1. Bound Fab was detected by staining the cells with secondary goat anti-human Fab–FITC conjugate and analyzing by FACS (black lines). Green lines indicate staining with the secondary antibody only.
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External Collaborators Zhen-qing Feng, Nanjing Medical University, China Milton Gross, Department of Veterans Affairs Medical Center/University of Michigan, Ann Arbor Xiao-hong Guan, Nanjing Medical University, China Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington Yi Ren, Cancer Center, Cleveland Clinic Foundation, Cleveland, Ohio Kang-lin Wan, Chinese Centers for Disease Control and Prevention, Beijing, China David Waters, Gerald P. Murphy Cancer Foundation, Seattle, Washington David Wenkert, Michigan State University, East Lansing Wei-cheng You, Beijing Institute for Cancer Research, China Recent Publications Jiao, Y., P. Zhao, J. Zhu, T. Grabinski, Z. Feng, X. Guan, R.S. Skinner, M.D. Gross, Y. Su, G.F. Vande Woude, R.V. Hay, and B. Cao. In press. Construction of human naïve Fab library and characterization of anti-Met Fab fragment generated from the library. Molecular Biotechnology. Zhang, Yu-Wen, Yanli Su, Nathan Lanning, Margaret Gustafson, Nariyoshi Shinomiya, Ping Zhao, Brian Cao, Galia Tsarfaty, Ling-Mei Wang, Rick Hay, and George F. Vande Woude. 2005. Enhanced growth of human Met-expressing xenografts in a new strain of immunocompromised mice transgenic for human hepatocyte growth factor/scatter factor. Oncogene 24(1): 101–106. Tan, Min-Han, Carl Morrison, Pengfei Wang, Ximing Yang, Carola J. Haven, Chun Zhang, Ping Zhao, Maria S. Tretiakova, Eeva Korpi-Hyovalti, John R. Burgess, Khee Chee Soo, Wei-Keat Cheah, Brian Cao, James Resau, Hans Morreau, and Bin Tean Teh. 2004. Loss of parafibromin immunoreactivity is a distinguishing feature of parathyroid carcinoma. Clinical Cancer Research 10(19): 6629–6637. Zhao, Ping, Xudong Liang, Jessica Kalbfleisch, Han-Mo Koo, and Brian Cao. 2003. Neutralizing monoclonal antibody against anthrax lethal factor inhibits intoxication in a mouse model. Human Antibodies 12(4): 129–135.
From left to right: Zhu, Wang, Cao, Ferrell, Grabinski, Zhao
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Laboratory of Mass Spectrometry and Proteomics Gregory S. Cavey, B.S. Mr. Cavey received his B.S. degree from Michigan State University in 1990. Prior to joining VARI he was employed at Pharmacia in Kalamazoo, Michigan, for nearly 15 years. As a member of a biotechnology development unit, he was group leader for a protein characterization core laboratory. More recently as a research scientist in discovery research, he was principal in the establishment and application of a stateof-the-art proteomics laboratory for drug discovery. Mr. Cavey joined VARI as a Special Program Investigator in July 2002. Laboratory Members
Staff Paula Davidson, M.S.
Student Wendy Johnson
Research Interests spectrometry data. We have optimized all aspects of this analysis for sample recovery yields and high-sensitivity protein identification.
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he Mass Spectrometry and Proteomics program works with many of the research labs at the Institute, using stateof-the-art mass spectrometers in combination with analytical protein separation and purification methods to help answer a wide range of biological questions. Using mass spectrometry data and database search software, proteins can be identified and characterized with unprecedented sensitivity and throughput. Since proteomics is a relatively new scientific discipline, many of the analytical techniques are rapidly changing; therefore our mission involves using established protocols, improving them, and developing new approaches to expand the scope of biological challenges being addressed.
Recently, we have been evaluating newly developed software that allows us to eliminate the electrophoresis separation step from these analyses, giving the potential to identify more proteins from complex mixtures. With this software, affinitypurified protein complexes are compared to a control sample via peptide differential display. The proteins are digested into peptides in solution rather than from gels and are analyzed by LC-MS. Peptides unique to the experimental sample relative to the control are used to identify proteins that are part of a protein complex. Protein characterization
Protein-protein interactions
Our laboratory also characterizes proteins and their post-translational modifications. Purified proteins are analyzed by protein electrospray to confirm the average protein molecular weight before proceeding to labor-intensive studies such as protein crystallization.
Analyzing samples representing different cellular conditions or disease states is a step toward understanding the role of a protein with an unknown function or understanding the regulatory mechanism of several proteins in a given pathway. In this approach, a known protein is affinitypurified from a nondenatured sample. The purified protein and its binding partners are separated using two-dimensional (2D) electrophoresis gels or SDS-PAGE. After staining, the proteins are cut from the gel, enzymatically digested into peptides, and then analyzed by nanoscale high-pressure liquid chromatography on line with a mass spectrometer (LC-MS). The mass spectrometer fragments the peptides and the resulting spectra are used to search protein or translated DNA databases. Identifications are made using the amino acid sequences derived from the mass
Mapping the post-translational modifications of proteins such as phosphorylation is an important undertaking in cancer research. Phosphorylation regulates many protein pathways, several of which could serve as potential drug targets for cancer therapy. In recent years, mass spectrometry has emerged as a primary tool that helps investigators determine exactly which amino acids of a protein are modified. This undertaking is complicated by many factors, but principally by the fact that pathway regulation can occur when only 0.01% of the molecules of a given protein are 16
from gels using mass spectrometry. Due to the labor-intensive nature of 2D gels and the underrepresentation of some classes of proteins (such as membrane proteins), proteomics has been moving toward solution-based separations and direct mass spectrometry. Our first approach is to digest all proteins into peptides and label their C-terminus with 18O water to effect a mass shift. Experimental and control samples are then mixed and separated by multidimensional high-pressure liquid chromatography using strong-cation ion exchange and reverse-phase separation modes. Peptides that are differentially expressed in experimental and control samples according to their 16O/18O ratio are identified using mass spectrometry and database searching.
phosphorylated. Thus, we are dealing with an extremely small number of molecules, in addition to the fact that the purification of phosphopeptides is always difficult. Our lab collaborates with investigators to map protein phosphorylation using techniques including immobilized metal affinity purification following esterification; immunoaffinity purification of phosphoproteins and peptides; and phosphorylation-specific mass spectrometry detection. Protein expression As mass spectrometry instruments and protein separation methods develop, we hope to identify and quantitate all the proteins expressed in a given cell or tissue, as a means of evaluating all of the physiological processes occurring within. This approach, termed systems biology, aims at understanding how all proteins interact to affect a biological outcome. Traditionally this approach has used 2D gel electrophoresis, image analysis of stained proteins, and identification of proteins
We intend to apply this or other mass spectrometry–based approaches in the discovery of biomarkers for early cancer detection, for morespecific diagnosis, and for more-accurate prognosis following drug treatment.
External Collaborators Greg Fraley, Hope College, Holland, Michigan Gary Gibson, Henry Ford Hospital, Detroit, Michigan Brett Phinney, Michigan State University, East Lansing Recent Publications Li, Yong, Mihwa Choi, Greg Cavey, Jennifer Daugherty, Kelly Suino, Amanda Kovach, Nathan C. Bingham, Steven A. Kliewer, and H. Eric Xu. 2005. Crystallographic identification and functional characterization of phospholipids as ligands for the orphan nuclear receptor steroidogenic factor-1. Molecular Cell 17(4): 491–502.
From left to right: Davidson, Johnson, Cavey
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Laboratory of Signal Regulation and Cancer Sara A. Courtneidge, Ph.D. Dr. Courtneidge completed her Ph.D. at the National Institute for Medical Research in London. She began her career in the basic sciences in 1978 as a postdoctoral fellow in the laboratory of J. Michael Bishop at the University of California School of Medicine. She later joined her alma mater as a member of the scientific staff. In 1985 Dr. Courtneidge joined the European Molecular Biology Laboratory as group leader and in 1991 was appointed a senior scientist with tenure. She joined Sugen in 1994 as Vice President of Research, later becoming Senior Vice President of Research and then Chief Scientist. Dr. Courtneidge joined VARI in January 2001 as a Distinguished Scientific Investigator. Laboratory Members
Staff Eduardo Azucena, Ph.D. Paul Bromann, Ph.D. Hasan Korkaya, Ph.D.
Ian Pass, Ph.D. Darren Seals, Ph.D. Laila Al-Duwaisan
Research Interests target of Src in vivo) and in normal cells after treatment with any of several growth factors. We recently found that in Src-transformed cells, Tks5/Fish is localized to specialized regions of the plasma membrane called podosomes (sometimes referred to as invadopodia). These actin-rich protrusions from the plasma membrane are sites of matrix invasion and locomotion. The PX domain of Tks5/Fish associates with phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, and it is required for targeting Tks5/Fish to podosomes. The fifth SH3 domain of Tks5/Fish mediates its association with members of the ADAMs family of membrane metalloproteases, which in Srctransformed cells are also localized to podosomes. We have begun to dissect the role of Tks5/Fish in Src-transformed cells with transformation. reduced Tks5/Fish levels no longer form podosomes and are poorly invasive. We detected Tks5/Fish expression in podosomes in invasive human cancer cell lines, as well as in tissue samples from human breast cancer and melanoma. Tks5/Fish expression was also required for invasion of human cancer cells. Furthermore, we have developed an assay to generate podosomes upon expression of Tks5/Fish, which will allow us to dissect the requirements for podosome formation in more detail. We are also investigating the potential of both Tks5/Fish and its binding proteins as markers of invasive disease and as potential therapeutic targets.
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ur laboratory wants to understand at the molecular level how proliferation is controlled in normal cells and by what mechanisms these controls are subverted in tumor cells. We largely focus on a family of oncogenic tyrosine kinases, the Src family. The prototype of the family, vSrc, originally discovered as the transforming protein of Rous sarcoma virus, is a mutated and activated version of a normal cellular gene product, cSrc. The activity of all members of the Src family is normally under strict control; however the enzymes are frequently activated or overexpressed, or both, in human tumors. In normal cells, Src family kinases (SFKs) have been implicated in signaling from many types of receptors, including receptor tyrosine kinases, integrin receptors, and G protein–coupled receptors. Signals generated by SFKs are thought to play roles in cell cycle entry, cytoskeletal rearrangements, cell migration, and cell division. In tumor cells, Src may play a role in growth factor–independent proliferation or in invasiveness. Furthermore, some evidence points to a role for SFKs in angiogenesis. Some of the current projects in the laboratory are outlined below. The role of the Src substrate Tks5/Fish in tumorigenesis Tks5/Fish is an adaptor protein which has five SH3 domains and a phox homology (PX) domain. Tks5/Fish is tyrosine-phosphorylated in Src-transformed fibroblasts (suggesting that it is a 18
The role of Src family kinases in mitogenic signaling pathways
promoted mRNA stabilization. We are currently exploring how SFKs signal gene expression by enhancing mRNA stability.
We have previously shown that Src family kinases are required for both Myc induction and DNA synthesis in response to platelet-derived growth factor (PDGF) stimulation of NIH3T3 fibroblasts. We have also previously identified and characterized a small-molecule inhibitor of Src family kinases called SU6656. We wanted to address whether there is a discrete SFK-specific pathway leading to enhanced gene expression, or whether SFKs act to generally enhance PDGFstimulated gene expression. To do this, we treated quiescent NIH3T3 cells with PDGF in the presence or absence of SU6656 and analyzed global patterns of gene expression. We determined that a discrete set of immediate early genes was induced by PDGF and inhibited by SU6656. We further determined that SFKs did not stimulate the rate of transcription of these genes, but rather
Breast cancer Increased Src activity can be demonstrated in the majority of breast cancers, both estrogendependent and estrogen-independent, yet the role of Src in breast tumorigenesis has not been fully established. We have been characterizing the role of Src in estrogen-stimulated signal transduction pathways in breast cancer cell lines. We have shown that Src family kinase activity is required for estrogen to stimulate mitogenesis in MCF7 cells. Furthermore, inhibition of Src prevents estrogen stimulation both of Myc and of MAP kinase activity. We are currently dissecting which Src signaling pathways are necessary for estrogenstimulated growth, as well as how Src activity results in the activation of MAP kinase and in the production of Myc.
Recent Publications Bromann, Paul A., Hasan Korkaya, Craig P. Webb, Jeremy Miller, Tammy L. Calvin, and Sara A. Courtneidge. 2005. Platelet-derived growth factor stimulates Src-dependent mRNA stabilization of specific early genes in fibroblasts. Journal of Biological Chemistry 280(11): 10253–10263. Seals, Darren F., Eduardo F. Azucena, Jr., Ian Pass, Lia Tesfay, Rebecca Gordon, Melissa Woodrow, James H. Resau, and Sara A. Courtneidge. 2005. The adaptor protein Tks5/Fish is required for podosome formation and function, and for the protease-driven invasion of cancer cells. Cancer Cell 7(2): 155–165. Bromann, Paul A., Hasan Korkaya, and Sara A. Courtneidge. 2004. The interplay between Src family kinases and receptor tyrosine kinases. Oncogene 23(48): 7957–7968.
From left to right: Azucena, Seals, Pass, Al-Duwaisan, Bromann
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Laboratory of Cancer and Developmental Cell Biology Nicholas S. Duesbery, Ph.D. Dr. Duesbery received both his M.Sc. (1990) and Ph.D. (1996) degrees in zoology from the University of Toronto, Canada, under the supervision of Yoshio Masui. Before his appointment as a Scientific Investigator at VARI in April 1999, he was a postdoctoral fellow in the laboratory of George Vande Woude in the Molecular Oncology Section of the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute–Frederick Cancer Research and Development Center, Maryland. Laboratory Members
Staff Paul Spilotro, M.D. Philippe Depeille, Ph.D. Hilary Wagner, M.S. John Young, M.S. Elissa Boguslawski
Students Chia-Shia Lee Lisa Orcasitas
Visiting Scientist Gustavo Nacheli, M.D.
Research Interests The therapeutic potential of anthrax lethal toxin
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he overall goal of the Laboratory of Cancer and Developmental Cell Biology is to further the study of mitogenactivated protein kinase kinase (MEK) signaling in health and disease. Currently, work performed in the lab is organized into three projects to explore 1) MEK signaling and tumor biology, 2) the therapeutic potential of anthrax lethal toxin (LeTx), and 3) molecular mechanisms of LeTx action.
Data from the National Cancer Institute’s Anti-Neoplastic Drug Screen indicates that several tumor types, notably melanomas and colorectal adenocarcinomas, are sensitive to LeTx. In addition, we have noted that angio-proliferative tumors are also very sensitive to LeTx treatment. Consequently we have undertaken a systematic evaluation of the effects of LeTx upon human tumor-derived melanomas, colorectal adenocarcinomas, and Kaposi’s sarcoma. The goal of this project is to develop novel therapeutic agents that may be efficacious in the treatment of human malignancies. In 2004 we successfully obtained funding from the National Institutes of Health to evaluate the therapeutic potential of LeTx in the treatment of Kaposi’s sarcoma.
MEK signaling and tumor biology Many malignant sarcomas, such as angiosarcomas, are refractory to currently available treatments. However, sarcomas possess unique vascular properties which indicate they may be more responsive to therapeutic agents that target endothelial function. MEKs have been demonstrated to play an essential role in the growth and vascularization of carcinomas. We hypothesize that signaling through multiple MEK pathways is also essential for growth and vascularization of sarcomas. The objective of this research is to define the role of MEK signaling in the growth and vascularization of human sarcoma and to determine whether inhibition of multiple MEKs by agents such as anthrax lethal toxin, a proteolytic inhibitor of MEKs, may form the basis of a novel therapeutic approach to the treatment of human sarcoma. In 2004 we successfully obtained funding from the National Institutes of Health for this project.
Molecular mechanisms of LeTx action The lethal effects of Bacillus anthracis have been attributed to an exotoxin that it produces. This exotoxin is composed of three proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). EF is an adenylate cyclase and together with PA forms a toxin referred to as edema toxin. LF is a Zn2+-metalloprotease which together with PA forms a toxin referred to as lethal toxin. LeTx is the dominant virulence factor produced by B. anthracis and is the major cause of death in infected animals. The goal of this project is to develop a detailed molecular understanding of 20
Spilotro, who joined us in August. Paul is currently evaluating MEK signaling in human fibrosarcoma. Elissa Boguslawski and Lisa Orcasitas joined our team in September. Elissa will serve as our vivarium technician in charge of murine studies, including xenografts. Lisa is a Bridges to the Baccalaureate student and is currently making sarcoma tissue microarrays so that she can evaluate MEK signaling in human tumor samples. In the summer of 2004, the lab was joined by Mia Hemmes, an undergraduate student from Michigan State University, who undertook a cytogenetic analysis of a primary human sarcoma-derived cell line. As well, we hosted Ricky Gonzalez and Lynda Gladding, two Grand Rapids area high school students interested in careers in biological research. Ricky and Lynda evaluated the sensitivity of murine endothelial cells to LeTx.
the LF/MEK interaction that will facilitate the development of therapeutic agents for anthrax. In 2004, we identified a cluster of surface-exposed residues of LF that are distal to the catalytic site and are essential for its catalytic activity (Fig. 1). Details of this study were published in the Journal of Biological Chemistry. Staff notes Sherrie Boone, who has served as a technician in the lab since 2001, has left us. She was replaced in February 2004 by John Young, an M.Sc. graduate from the University of Oregon. John has initiated studies of LeTx and Kaposi’s sarcoma and has played a significant role in our identification of novel regions of LF that are required for its activity. Xudong Liang, a postdoctoral fellow who joined us in 2002, has moved on to a new position at the University of Minnesota. In his place we welcome Paul
Figure 1. A surface plot of anthrax LF highlighting mutagenized residues. A space-filled surface plot of LF was generated using Protein Explorer freeware. Residues identified as being critical for LF activity are colored yellow (K294), green (L293), red (L514), purple (N516), and orange (R491). Residues found to play a neutral or marginal role in LF activity are white. The NH2-terminus of MEK is indicated in black. A magnified image of this region shows that the critical residues are organized side-by-side in a focused band (KLLNR), which lies at one end of the catalytic groove.
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External Collaborators Jean-François Bodart, Université des Sciences et Technologies de Lille, France. Art Frankel, Wake Forest University, Winston-Salem, North Carolina Silvio Gutkind, National Institute of Dental and Craniofacial Research, Bethesda, Maryland Stephen Leppla, National Institute of Allergies and Infectious Diseases, Bethesda, Maryland Recent Publications Bodart, J.-F., and N.S. Duesbery. In press. Xenopus tropicalis oocytes: more than just a beautiful genome. In Cell Biology and Signal Transduction, J. Liu, ed. Humana Press. Singh, Yogendra, Xudong Liang, and Nicholas S. Duesbery. 2005. Pathogenesis of Bacillus anthracis: the role of anthrax toxins. In Microbial Toxins: Molecular and Cellular Biology, T. Proft, ed. Norwich, U.K.: Horizon Scientific, pp. 285–312. Liang, Xudong, John J. Young, Sherrie A. Boone, David S. Waugh, and Nicholas S. Duesbery. 2004. Involvement of domain II in toxicity of anthrax lethal factor. Journal of Biological Chemistry 279(50): 52473–52478.
From left to right, standing: Lee, Duesbery, Depeille, Young, Cumbo-Nacheli, Spilotro seated: Holman, Boguslawski, Wagner
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Microinjection of embryonic stem cells In these photos, technicians are microinjecting embryonic stem cells into mouse blastocysts for gene targeting studies. (Photos by Julie Koeman)
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Vivarium and Laboratory of Transgenics Bryn Eagleson, A.A., RLATG Bryn Eagleson began her career in laboratory animal services in 1981 with Litton Bionetics at the National Cancer Institute’s Frederick Cancer Research and Development Center (NCI-FCRDC) in Maryland. In 1983, she joined the Johnson & Johnson Biotechnology Center in San Diego, California. In 1988, she returned to the NCI-FCRDC, where she continued to develop her skills in transgenic technology and managed the transgenic mouse colony. During this time Ms. Eagleson attended Frederick Community College and Hood College in Frederick, Maryland. In 1999, she joined VARI as the Vivarium Director and Transgenics Special Program Manager. Laboratory Members
Managerial staff Jason Martin, RLATG
Technical staff Dawna Dylewski, B.S. Audra Guikema, B.S., L.V.T. Elissa Boguslawksi, RALAT Jamie Bondsfield Sylvia Marinelli
Vivarium staff Lisa DeCamp, B.S. Laura Sixburry, B.S. Crystal Brady Kathy Geil Jarred Grams Tina Schumaker
Research Interests genetic material and one derived from the sperm that contains the paternal genetic material. As development proceeds, these two pronuclei fuse, the genetic material mixes, and the cell proceeds to divide and develop into an embryo. Transgenic mice are produced by injecting small quantities of foreign DNA (the transgene) into a pronucleus of a one-cell fertilized egg. DNA microinjected into a pronucleus randomly integrates into the mouse genome and will theoretically be present in every cell of the resulting organism. Expression of the transgene is controlled by elements called promoters that are genetically engineered into the transgenic DNA. Depending on the selection of the promoter, the transgene can be expressed in every cell of the mouse or in specific cell populations such as neurons, skin cells, or blood cells. Temporal expression of the transgene during development can also be controlled by genetic engineering. These transgenic mice are excellent models for studying the expression and function of the transgene in vivo.
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he goal of the vivarium and the transgenics laboratory is to develop, provide, and support high-quality mouse modeling services for the Van Andel Research Institute investigators, Michigan Technology TriCorridor collaborators, and the greater research community. We use two Topaz Technologies software products, Granite and Scion, for integrated management of the vivarium finances, the mouse breeding colony, and the Institutional Animal Care and Use Committee (IACUC) protocols and records. Imaging equipment, such as the PIXImus mouse densitometer and the Acuson Sequoia 512 ultrasound machine, is available for noninvasive imaging of mice. VetScan blood chemistry and hematology analyzers are now available for blood analysis. Also provided by the vivarium technical staff are an extensive xenograft model development and analysis service, rederivation, surgery, dissection, necropsy, breeding, and health-status monitoring. Transgenics Fertilized eggs contain two pronuclei, one that is derived from the egg and contains the maternal
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Standing from left to right: Bondsfield, Sixbury, Grams, DeCamp, Brady, Guikema, Martin seated from left to right: Schumaker, Dylewski, Marinelli, Eagleson, Boguslawski
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Bioinformatics Special Program Kyle A. Furge, Ph.D. Dr. Furge received his Ph.D. in biochemistry from the Vanderbilt University School of Medicine in 2000. Prior to obtaining his degree, he worked as a software engineer at YSI, Inc., where he wrote operating systems for embedded computer devices. Dr. Furge did his postdoctoral work in the laboratory of Dr. George Vande Woude and became a Bioinformatics Scientist at VARI in June of 2001. Laboratory Members
Staff Karl Dykema, B.A.
Research Interests Technology Laboratory in the placement of quality control markers on gene expression microarrays. These microarrays contain more than 20,000 unique DNA fragments that are robotically placed on a small glass slide. In order to ensure the DNA fragments are placed correctly, the quality control markers are robotically placed on the arrays in a very specific pattern as the arrays are constructed. As each array is produced, a quick visual inspection of the pattern of quality control markers can be used to verify that all the DNA fragments were placed on the arrays correctly.
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s high-throughput biotechnologies such as DNA sequencing, gene expression microarrays, and genotyping become more available to researchers, analysis of the data produced by these technologies becomes Disciplines such as increasingly difficult. bioinformatics and computational biology have recently emerged to help develop methods that assist in the storage, distribution, integration, and analysis of these data sets. The bioinformatics program at VARI is currently focused on using computational approaches to understand how cancer cells differ from normal cells at the molecular level. In addition, VARI is also part of the overall bioinformatics effort in the state of Michigan through the Michigan Center for Biological Information.
In addition to assisting other VARI research labs, our group has a special focus on understanding how cytogenetic abnormalities influence cancer development and progression. Many cancer types, including liver and kidney cancers, are associated with defined sets of DNA gains and losses. For example, the majority of hepatocellular carcinomas contain extra copies of chromosome 1p and lack copies of chromosome 4q. In contrast, the majority of clear cell renal cell carcinomas contain an extra copy of chromosome 5q and lack a copy of chromosome 3p. Interestingly, we and other groups have noticed that transcription is dramatically disrupted within regions of DNA copy number change. We are currently developing and testing a number of different algorithms to identify these disrupted regions using gene expression data. In addition, we are developing methods to identify key regulatory genes that lie within the abnormal region and may be involved in tumor development and/or progression.
Laboratory members from the bioinformatics program have worked on a wide variety of projects to further the research efforts at VARI in 2004. Recently, we constructed a program to identify short sequences within genes that are likely to be responsive to siRNA interference. siRNAs are short, double-stranded DNA sequences that when introduced into living cells bind to the RNA produced from a gene of interest and inhibit expression of the gene. As such, the introduction of siRNAs is becoming a more widely used technique to examine the role of individual genes in both cancerous and noncancerous cells. The program we developed contained a new algorithm, developed by one of the VARI investigators, to identify potential sites within genes that would be sensitive to siRNAs. In another project, we assisted the Microarray
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External Collaborators Xin Chen, Stanford University, Stanford, California Recent Publications Yang, X.J., M.-H. Tan, H.L. Kim, J.A. Ditlev, M.W. Betten, C.E. Png, E.J. Kort, K. Futami, K.J. Dykema, K.A. Furge, M. Takahashi, H. Kanayama, P.H. Tan, B.S. Teh, C. Luan, et al. In press. A molecular classification of papillary renal cell carcinoma. Cancer Research. Dykema, K.J., and K.A. Furge. 2004. Diminished transcription of chromosome 4q is inversely related to local spread of hepatocellular carcinoma. Genes, Chromosomes and Cancer 41(4): 390–394. Furge, Kyle A., Kerry A. Lucas, Masayuki Takahashi, Jun Sugimura, Eric J. Kort, Hiro-omi Kanayama, Susumu Kagawa, Philip Hoekstra, John Curry, Ximing J. Yang, and Bin T. Teh. 2004. Robust classification of renal cell carcinoma based on gene expression data and predicted cytogenetic profiles. Cancer Research 64(12): 4117–4121. Haven, C.J., V.M. Howell, P.H.C. Eilers, R. Dunne, M. Takahashi, M. van Puijenbroek, K. Furge, J. Kievit, M.-H. Tan, G.J. Fleuren, B.G. Robinson, L.W. Delbridge, J. Philips, A.E. Nelson, U. Krause, et al. 2004. Gene expression of parathyroid tumors: molecular subclassification and identification of the potential malignant phenotype. Cancer Research 64(20): 7405–7411. Lindvall, Charlotta, Kyle Furge, Magnus Björkholm, Xiang Guo, Brian Haab, Elisabeth Blennow, Magnus Nordenskjöld, and Bin Tean Teh. 2004. Combined genetic and transcriptional profiling of acute myeloid leukemia with normal and complex karyotypes. Haematologica 89(9): 1072–1081. Sugimura, Jun, Richard S. Foster, Oscar W. Cummings, Eric J. Kort, Masayuki Takahashi, Todd T. Lavery, Kyle A. Furge, Lawrence H. Einhorn, and Bin Tean Teh. 2004. Gene expression profiling of earlyand late-relapse nonseminomatous germ cell tumor and primitive neuroectodermal tumor of the testis. Clinical Cancer Research 10(7): 2368–2378. Tan, Min-Han, Craig G. Rogers, Jeffrey T. Cooper, Jonathon A. Ditlev, Thomas J. Maatman, Ximing Yang, Kyle A. Furge, and Bin Tean Teh. 2004. Gene expression profiling of renal cell carcinoma. Clinical Cancer Research 10(18): 6315S–6321S. Yang, Ximing J., Jun Sugimura, Maria S. Tretiakova, Kyle Furge, Gregory Zagaja, Mitchell Sokoloff, Michael Pins, Raymond Bergan, David J. Grignon, Walter M. Stadler, Nicholas J. Vogelzang, and Bin Tean Teh. 2004. Gene expression profiling of renal medullary carcinoma: potential clinical relevance. Cancer 100(5): 976–985.
From left to right: Furge, Dykema
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Laboratory of Cancer Immunodiagnostics Brian B. Haab, Ph.D. Dr. Haab obtained his Ph.D. in chemistry from the University of California at Berkeley in 1998. He then served as a postdoctoral fellow in the laboratory of Patrick Brown in the Department of Biochemistry at Stanford University. Dr. Haab joined VARI as a Special Program Investigator in May 2000. Laboratory Members
Staff Songming Chen, Ph.D. Michael Shafer, Ph.D. Sara Forrester, B.S. Darren Hamelinck, B.S. Randall Orchekowski, B.S.
Students Thomas LaRoche Richard Schildhouse
Research Interests makes use of gel and chromatographic separations and mass spectrometry to provide complementary experimental information.
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any cancers are difficult to detect at early stages and are often diagnosed too late to allow curative treatment. Earlier and more accurate detection of cancer could lead to better outcomes for many patients. A greater knowledge of the molecular changes associated with the development of cancer could lead to a better understanding of disease mechanisms and improved diagnostic tests. The Haab laboratory is developing novel experimental approaches to gather such molecular information and to use it for the diagnosis of cancer. Our goal is that these studies will produce measurable benefits to cancer patients.
An ongoing study of protein profiles from the sera of pancreatic cancer patients and controls (in collaboration with Randall Brand and George Vande Woude) shows the value of antibody microarrays for diagnostics research. Protein profiles from antibody microarrays targeting a wide variety of proteins—such as those previously associated with cancer, involved in biological systems altered by cancer, or having elevated levels in the tumor environment—are revealing many proteins at either higher or lower abundances in the cancer patients. Some of the proteins were not known to be associated with pancreatic cancer and may contribute to improved early detection of the disease. The serum samples can be classified as from cancer or control patients using the antibody measurements. The accuracy of the classifications was greatly improved with multiple measurements in combination (relative to using individual measurements); we need to further develop this approach for cancer diagnostics.
We are taking a variety of approaches to identify changes in blood protein composition that define cancer and that could be diagnostically useful. Microarray methods have features that are particularly useful for this research. We have been developing several related antibody and protein microarray methods—such as two-color competition assays, sandwich assays, glycan detection, and antigen detection of antibodies—to analyze serum samples from cancer patients and controls. With these methods (Fig. 1), we can efficiently probe the binding to many different antibodies and proteins and explore the use of multiple measurements for classifying samples. Previous technological developments that are now in routine use include a high-sensitivity detection method (two-color rolling-circle amplification) and a new method for isolating multiple microarrays on a single slide, allowing highthroughput sample processing. Our research also
A recent modification to this technology measures alterations in the glycosylation state of the proteins. Glycosylation—the attachment of specific carbohydrate structures to proteins— plays a major role in determining protein function, and glycosylation alterations have been associated with the development and progression of cancer. The ability to conveniently measure changes in specific carbohydrates on different proteins could be valuable in identifying the changes most 28
associated with cancer and thus of use for diagnostics. We have methods for detecting changes in glycosylation on proteins captured by antibody microarrays (see Fig. 1C) and are using those methods to profile the glycosylation alterations on serum proteins from cancer patients and control subjects. We use protein microarrays (see Fig. 1D) to gather additional information about changes occurring in the blood of cancer patients. Some tumor proteins elicit the production of antibodies targeting those proteins. The identification and measurement of tumor-recognizing antibodies could provide information about molecular alterations in the tumor and be valuable in cancer detection. The protein microarray is an ideal screening tool for that purpose. In collaboration with Gilbert Omenn and Samir Hanash, microarrays of tumor-derived proteins from cancer cell lines are probed with sera from cancer patients to identify proteins recognized by the patients’ antibodies. An ongoing study of serum from prostate cancer patients (in collaboration with Alan Partin) has identified several proteins that could be involved in an immune response. We are characterizing the nature of the responses and the proteins involved.
Figure 1. Antibody and protein microarray formats. A) Two-color competition assay. Two pools of proteins, respectively labeled with biotin and digoxigenin tags, are mixed and co-incubated on antibody microarrays. The relative amount of binding to each antibody from the two pools is determined through detection of the biotin and digoxigenin tags. B) Sandwich assay. After incubation of a protein sample on an antibody microarray, the amount of protein binding to each antibody is measured using a second antibody that targets the captured proteins. C) Glycan detection. A pool of digoxigenin-labeled proteins is incubated on antibody microarrays, and the level of protein binding at each antibody is determined by detection of the digoxigenin tag. The amount of a particular glycan on the captured proteins is detected using a biotin-labeled protein that specifically binds to that glycan. D) Protein array detection of antibodies. Serum samples are incubated on arrays containing a variety of tumor-derived proteins. Antibodies in the serum samples that recognize and bind to the proteins are detected using a secondary antibody that binds to human antibodies.
The above methods may be applied in a novel way to further their effectiveness. We have begun to develop and improve existing diagnostic markers through the use of longitudinal measurements (serial measurements over time). In a collaboration with William Catalona, Robert Vessella, and Ziding Feng, we are looking at changes over time in the concentrations of several serum proteins leading up to disease recurrence in prostate cancer patients. We hypothesize that the use of individualized thresholds defining abnormal protein levels, defined by each person’s history of measurements, will yield improved diagnostic accuracy over the use of single, population-wide thresholds. Our hypothesis has been supported in some individual demonstrations, and we now have an experimental system for systematically exploring it for a large number of proteins and many patients.
We are seeking to apply the methods described above in ways that will have a positive result in terms of cancer care. The incorporation of mass spectrometry methods, through collaborations with Greg Cavey at VARI and members of the Michigan Proteome Consortium, will provide more opportunities for discovery. The further testing of the value of these tools is being pursued through local clinical oncology programs. The access to clinical practice is especially valuable for translating the development and discovery in our laboratory into benefits for cancer patients.
External Collaborators Phil Andrews, Gilbert Omenn, and Diane Simeone, University of Michigan, Ann Arbor Randall Brand, Evanston Northwestern Healthcare, Illinois E. Brian Butler and Bin S. Teh, Baylor College of Medicine, Houston, Texas William Catalona, John Grayhack, and Anthony Schaeffer, Northwestern University, Evanston, Illinois 29
Jose Costa and Paul Lizardi, Yale University School of Medicine, New Haven, Connecticut Deborah Dillon, Brigham and Women’s Hospital, Boston, Massachusetts Ziding Feng and Samir Hanash, Fred Hutchinson Cancer Research Center, Seattle, Washington Jorge Marrero, University of Michigan Hospital, Ann Arbor Alan Partin, Johns Hopkins University, Baltimore, Maryland Peter Schirmacher, University of Cologne, Germany Robert Vessella, University of Washington, Seattle Cornelius Verweij, University of Amsterdam, The Netherlands Recent Publications Hamelinck, D., H. Zhou, L. Li, Z. Feng, C. Verweij, D. Dillon, J. Costa, and B.B. Haab. In press. “Optimized normalization for antibody microarrays and the identification of serum protein alterations associated with pancreatic cancer.” Molecular & Cellular Proteomics. Haab, B.B., and P.M. Lizardi. In press. “RCA-enhanced protein detection arrays.” Methods in Molecular Biology. Breuhahn, Kai, Sebastian Vreden, Ramsi Haddad, Susanne Beckebaum, Dirk Stippel, Peer Flemming, Tanja Nussbaum, Wolfgang H. Caselmann, Brian B. Haab, and Peter Schirmacher. 2004. Molecular profiling of human hepatocellular carcinoma defines mutually exclusive interferon regulation and insulin-like growth factor II overexpression. Cancer Research 64(17): 6058–6064. Konwinski, R., R. Haddad, J.A. Chun, S. Klenow, S. Larson, B.B. Haab, and L.L. Furge. 2004. Oltipraz, 3H-1,2-dithiole-3-thione and sulforaphane induce overlapping and protective antioxidant responses in murine microglial cells. Toxicology Letters 153(3): 343–355. Lindvall, Charlotta, Kyle Furge, Magnus Björkholm, Xiang Guo, Brian Haab, Elisabeth Blennow, Magnus Nordenskjöld, and Bin Tean Teh. 2004. Combined genetic and transcriptional profiling of acute myeloid leukemia with normal and complex karyotypes. Haematologica 89(9): 1072–1081. Qiu, Ji, Juan Madoz-Gurpide, David E. Misek, Rork Kuick, Dean E. Brenner, George Michailidis, Brian B. Haab, Gilbert S. Omenn, and Sam Hanash. 2004. Development of natural protein microarrays for diagnosing cancer based on an antibody response to tumor antigens. Journal of Proteome Research 3(2): 261–267. Zhou, Heping, Kerri Bouwman, Mark Schotanus, Cornelius Verweij, Jorge A. Marrero, Deborah Dillon, Jose Costa, Paul Lizaardi, and Brian B. Haab. 2004. Two-color, rolling-circle amplification on antibody microarrays for sensitive, multiplexed serum-protein measurements. Genome Biology 5(4): R28.
From left to right: Haab, Hamelinck, Orchekowski, Chen, Shafer, Forrester
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Molecular Medicine and Virology Group Sheri L. Holmen, Ph.D. Dr. Holmen received her M.S. in biomedical science from Western Michigan University in 1995 and her Ph.D. in tumor biology from the Mayo Clinic College of Medicine in 2000. She did her postdoctoral work at VARI in the laboratory of Bart Williams from 2000 to 2003 and became a Junior Investigator at VARI in December 2003. Laboratory Members
Staff Marleah Russo
Research Interests he primary focus of the Molecular Medicine and Virology Group is to identify molecules that can be effective targets for cancer therapy, with the goal of developing better therapies with fewer side effects. The sequencing of the human genome has yielded a wealth of biological data, but we still know relatively little about which genes are causally associated with tumor development and which are only markers of cancer. Because of the high cost of developing new therapies, it is important that we identify which genetic changes can be productively targeted. We are concentrating our initial efforts on melanoma and glioblastoma, tumors which demonstrate constitutive activation of Ras signaling.
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These vectors are termed RCASBP or RCANBP. The ability of these vectors to infect non-avian cells relies on expression of the corresponding receptor on the cell surface. The viral receptor is typically introduced into mammalian cells (or mice) via an inducible and/or tissue-specific transgene. Therefore, this system allows for tissueand cell-specific targeted infection of mammalian cells through ectopic expression of the viral receptor. Alternatively, when targeted infection of mammalian cells is not required (e.g., in cell culture), infection can be achieved through the use of non-avian envelopes, such as the amphotropic envelope from murine leukemia virus. The receptor for this envelope is endogenously expressed on almost all mammalian cells.
The RCAS system
We have used the RCASBP/RCANBP family of retroviral vectors extensively in both cultured cells and live animals for studies of viral replication and of cancer modeling in mice. Most of these studies have analyzed gain-of-function phenotypes by delivering and overexpressing a particular gene of interest. Recently, we engineered the RCANBP vector to reduce the expression of specific genes through the delivery of short hairpin RNA sequences. We also engineered this vector to control the expression of the inserted gene using the tetracycline (tet)regulated system. Sequences inserted into this region are transcribed from a tet-responsive element and not the viral LTR. This virus allows inserted genes to be turned on and off in order to determine if expression of the gene is required for tumor initiation, maintenance, and progression. The ability to turn off gene expression will help determine if that gene is a good target for therapy.
We use a series of replication-competent retroviral vectors based on the SR-A strain of Rous sarcoma virus (RSV), a member of the avian leukosis virus (ALV) family, to study the roles of different genes in tumor initiation and progression. RSV is the only known naturally occurring, replication-competent retrovirus that carries an oncogene, src. In the RCAS vectors, the region encoding src (which is dispensable for viral replication) has been replaced by a synthetic DNA linker. Foreign genes inserted into this linker are expressed from the viral LTR promoter via a subgenomic splice site (just as src is in RSV). RCAN vectors differ from RCAS vectors in that they lack the src splice acceptor; the gene of interest is inserted along with an internal promoter. Higher-titer viruses subsequently have been generated by replacing the RSV SR-A pol gene with the pol gene of the Bryan strain of RSV. 31
Melanoma
deficient mice results in the development of multiple spontaneous cutaneous melanomas. However, unlike in the human disease, these tumors fail to metastasize. A conditional Ink4a/Arf knockout allele, Ink4a-lox, has been introduced into the germline of FVB/N mice. The lox sites flank exons 2 and 3 of this locus such that Cremediated excision eliminates both p16INK4A and p19ARF. We have recently obtained two homozygous Ink4a-lox/lox breeding pairs, which will be crossed to our TRP2-TVA transgenic mice. We plan to use the mice to study the role of different genes in melanoma initiation, maintenance, and progression.
Melanoma is the most rapidly increasing malignancy among young people in the United States. If detected early, the disease is easily treated, but once the disease has metastasized it has a high mortality rate. Current systemic therapy for advanced, metastatic melanoma includes dacarbazine (DTIC) chemotherapy, either alone or in combination with other agents, and biological therapy using recombinant interferon-Îą (IFN-Îą) and/or interleukin-2 (IL-2). However, except on rare occasions, none of these treatments has produced long-term control of the disease, and cytokine therapy is associated with significant toxicities.
Glioblastoma
We have developed a mouse model of melanoma based on the avian RCAS/TVA system. In this model, the retroviral receptor TVA is expressed under the control of the tyrosinaserelated protein 2 (TRP2) promoter, which is expressed in melanocytes. In replicating mammalian cells that express TVA, the viral vector is capable of stably integrating into the DNA and expressing the experimental gene at high levels, but the virus is replication-defective because viral RNA and proteins are inefficiently produced. Therefore, the viral vectors cannot spread in the target animals; in addition, since little viral envelope protein is produced, there is no interference to superinfection. Theoretically, there is no limit to the number of experimental genes that can be introduced into a TVA-expressing mammalian cell. The ability of these cells to be infected by multiple viruses allows efficient modeling of melanoma, because multiple oncogenic alterations can be introduced into the same cell or animal without the costs associated with mating multiple strains of transgenic mice.
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. It is also the most fatal: mean survival is less than one year from the time of diagnosis, with less than 10% survival after two years. Despite major improvements in imaging, radiation, and surgery, the prognosis for patients with this disease has not changed in the last 20 years. Recently, genes that are differentially expressed in tumor tissue relative to normal brain tissue have been found. However, those that can be productively targeted for therapeutic intervention in human patients remain to be identified. A mouse model of human GBM based on the avian RCAS/TVA system has been developed by Eric Holland. In this model, the retroviral receptor TVA is expressed under the control of the Nestin promoter, which is active in neural and glial progenitors. Intracranial infection of Nestin-TVA mice with RCASBP(A)-Akt and RCASBP(A)KRas induces glioblastomas that are histologically similar to human GBM. We have generated an RCANBP(A)TRE-KRas virus in which expression of K-Ras can be controlled post-delivery. We are using the Nestin-TVA model to test the function of this virus in vivo. We plan to use these vectors to determine if K-Ras expression is required for tumor maintenance in this system and to identify which genes are appropriate targets for therapy.
Activated ras oncogenes, which turn on mitogen-activated protein kinase (MAPK) signaling, are detected in approximately 20% of human melanomas. Recently, activating mutations in the B-raf gene, which also activate MAPK signaling, have been found in more than 65% of malignant melanomas. With mutually exclusive mutations in ras and B-raf, the MAPK signaling pathway is constitutively activated in over 85% of cases of malignant melanoma, indicating its importance. Overexpression of activated H-ras (G12V) specifically in melanocytes of Ink4a-
Antiviral strategies A second, related focus of our group is the identification of effective antiviral strategies using the RCASBP/RCANBP family of retroviral vectors as a model system. Viral diseases pose a 32
viral life cycle. The results have provided valuable information on the biology of avian viruses, as well as having the potential for practical application. We are now working to adapt the new RNA interference (RNAi) technology to the development of new anti-viral strategies for two important chicken pathogens, ALV and Marek’s Disease virus (MDV). These two targets have been chosen because both are economically important and because they have distinctly different infectious cycles that provide different challenges for RNAi.
major risk to the food supply and to animal welfare, especially in today’s high-intensity animal agriculture. Many viruses are highly communicable and are capable of rapid mutation to escape immune surveillance; few effective antiviral drugs are available. Over the past several years, we have developed strategies aimed at conferring dominant resistance to viral pathogens in chickens. These strategies have focused on manipulating viral (“pathogen-derived resistance”) and/or cellular genes (“host-derived resistance”) to express new proteins capable of disrupting the
External Collaborators Jerry Dodgson, Michigan State University, East Lansing Henry Hunt and Huanmin Zhang, Avian Disease and Oncology Laboratory, East Lansing, Michigan Recent Publications Ai, Minrong, Sheri L. Holmen, Wim van Hul, Bart O. Williams, and Matthew W. Warman. 2005. Reduced affinity to and inhibition by DKK1 form a common mechanism by which high bone mass–associated missense mutations in LRP5 affect canonical Wnt signaling. Molecular and Cellular Biology 25(12): 4946–4955. Holmen, Sheri L., Scott A. Robertson, Cassandra R. Zylstra, and Bart O. Williams. 2005. Wnt-independent activation of ß-catenin mediated by a Dkk-1-Frizzled 5 fusion protein. Biochemical and Biophysical Research Communications 328(2): 533–539. Holmen, Sheri L., Cassandra R. Zylstra, Aditi Mukherjee, Robert Sigler, Marie-Claude Faugere, Mary Bouxsein, Lianfu Deng, Thomas Clemens, and Bart O. Williams. 2005. Essential role of ß-catenin in postnatal bone acquisition. Journal of Biological Chemistry 280(22): 21162–21168. Sanchez-Perez, Luis, Timothy Kottke, Rosa Maria Diaz, Atique Ahmed, Jill Thompson, Heung Chong, Alan Melcher, Sheri Holmen, Gregory Daniels, and Richard G. Vile. 2005. Potent selection of antigen loss variants of B16 melanoma following inflammatory killing of melanocytes in vivo. Cancer Research 65(5): 2009–2017. Bromberg-White, Jennifer L., Craig P. Webb, Veronique S. Patacsil, Cindy K. Miranti, Bart O. Williams, and Sheri L. Holmen. 2004. Delivery of short hairpin RNA sequences by using a replication-competent avian retroviral vector. Journal of Virology 78(9): 4914–4916. Holmen, Sheri L., Troy A. Giambernardi, Cassandra R. Zylstra, Bree D. Buckner-Berghuis, James H. Resau, J. Fred Hess, Vaida Glatt, Mary L. Bouxsein, Minrong Ai, Matthew L. Warman, and Bart O. Williams. 2004. Decreased BMD and limb deformities in mice carrying mutations in both Lrp5 and Lrp6. Journal of Bone and Mineral Research 19(12): 2033–2040. From left to right: Russo, Holmen
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Laboratory of Integrin Signaling and Tumorigenesis Cindy K. Miranti, Ph.D. Dr. Miranti received her M.S. in microbiology from Colorado State University in 1982 and her Ph.D. in biochemistry from Harvard Medical School in 1995. She was a postdoctoral fellow in the laboratory of Dr. Joan Brugge at ARIAD Pharmaceuticals, Cambridge, Mass., from 1995 to 1997 and in the Department of Cell Biology at Harvard Medical School from 1997 to 2000. Dr. Miranti joined VARI as a Scientific Investigator in January 2000. She is also an Adjunct Assistant Professor in the Department of Physiology at Michigan State University. Laboratory Members
Staff Suganthi Chinnaswamy, Ph.D. Mathew Edick, Ph.D. Robert Long, B.A. Veronique V. Schulz, B.S.
Student Erik Freiter
Research Interests frequently diagnosed cancer in U.S. men and the second leading cause of cancer death in men. Eighty percent of human prostate tumors arise in the peripheral zone of the gland and are primarily confined to the intermediate basal and secretory epithelial cells. Patients who at the time of diagnosis have androgen-dependent and organ-confined prostate cancer are relatively easy to cure through radical prostatectomy or localized radiotherapy. However, patients with aggressive and metastatic disease have fewer options. Androgen ablation can significantly reduce the tumor burden in these patients, but the potential for relapse and the development of androgen-independent cancer is high. Currently there are no effective treatments for patients who reach this stage of disease.
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ur laboratory is interested in understanding the mechanisms by which integrin receptors, interacting with the extracellular matrix, regulate cell processes involved in the development of cancer. Using tissue culture models, biochemistry, molecular genetics, and mouse models, we are defining the cellular and molecular events of integrindependent adhesion and downstream signaling that are important in melanoma and prostate tumorigenesis and metastasis. Integrins are transmembrane proteins that serve as receptors for extracellular matrix (ECM) proteins. By interacting with the ECM, integrins stimulate intracellular signaling transduction pathways that regulate cell shape, proliferation, migration, survival, gene expression, and differentiation. Integrins do not act autonomously; they are involved in “crosstalk” with receptor tyrosine kinases (RTKs) to regulate many cellular processes. Studies in our lab, for example, indicate that integrin-mediated adhesion to ECM proteins activates the epidermal growth factor receptors EGFR and ErbB2 and the HGF/SF receptor Met. Integrin-mediated activation of these RTKs is ligand-independent and is required for activation of a subset of intracellular signaling molecules in response to cell adhesion.
In the human prostate secretory glands, basal epithelial cells form a contiguous layer adjacent to the basement membrane. Upon them rests a layer of secretory luminal cells, forming a stratified epithelium. The basal cells express a broad repertoire of integrins, including α2, α3, α6, β1, and β4. The secretory cells express primarily α6 and β1, with some α2. In vivo, basal cells secrete and organize a laminin 5–containing basement membrane that also contains collagens IV and VII and laminin 10. The basal cells bind to laminin 5 and collegen VII through α6β4 to form hemidesmosomal complexes at the basal surface. Basal cells adhering to this matrix respond to growth factors secreted by the surrounding stroma, including EGF and HGF. They proliferate and give rise to the nonproliferating secretory cells through
The prostate gland and cancer Tumors that develop in cells of epithelial origin, i.e., carcinomas, represent the largest tumor burden in the United States. Prostate cancer is the most 34
as Ras/Erk signals generated by EGFR, are driving cell survival in primary cells (Fig. 1). We are currently exploring the downstream events that Src and Erk regulate to maintain cell survival on ECM.
the generation of an intermediate, transiently amplifying cell population that has traits of both cell types. In primary prostate tumors, α6β4 integrin and its ligands, laminin 5 and collagen VII, are lost. The tumor cells, unlike normal secretory cells, develop the ability to adhere, via α2β1 and α6β1, to an altered basement membrane consisting of collagen IV and laminin 10. Thus, tumor cells have characteristics of both basal and secretory cells, but not all the properties of either. Whether the tumor cells are derived from the transient differentiating population of basal cells or from differentiated secretory cells has not been unequivocally determined, but it is clear that the way in which these cells interact with the ECM has been changed. If tumor cells are derived from basal cells, they are now interacting with an altered matrix and using different integrins to engage it. If they are derived from the secretory cells, the tumor cells are now engaging a matrix, which they did not do previously. A fundamental question in our lab is whether the changes in integrin/matrix interactions that occur in tumor cells are required for or help to drive the survival of tumor cells.
Figure 1. Met and EGFR both act independently to regulate integrin-mediated survival of primary prostate epithelial cells. Activation of Src and Erk through Met and EGFR, respectively, are proposed to be involved in integrin-mediated survival.
We are also exploring the mechanisms by which integrins activate EGFR and ErbB2. This activation is ligand independent, requires only the cytoplasmic domain of EGFR, and stimulates the phosphorylation of only a subset of sites on EGFR. ErbB2 activation depends on EGFR, suggesting that integrins induce the formation of an EGFR/ErbB2 heterodimer. Integrin activation of Src or FAK is not required, but integrin activation of a phosphatase may be involved; we are currently investigating the role of several phosphatases.
The role of integrins and RTKs in prostate epithelial cell survival Increased cell survival due to resistance to cell death is a prerequisite for tumorigenesis. Several reports have suggested that the signaling pathways that regulate cell survival in normal prostate epithelial cells are different from those in prostate tumor cells. How integrin engagement of different ECMs regulates survival pathways is not known. We have recently found that integrin-induced activation of both EGFR and c-Met in primary prostate epithelial cells is required for cell survival in the absence of growth factors. Our goal is to determine how integrin activation of EGFR and c-Met regulate cell survival.
Integrin and RTK crosstalk in prostate cancer metastasis Death from prostate cancer is due to the development of metastatic disease, which is difficult to control. The mechanisms involved in progression to metastatic disease are not understood. Our approach is to characterize genes that are specifically associated with metastatic prostate cancer. CD82/KAI1 is a metastasis suppressor gene whose expression is specifically lost in metastatic cancer but not in primary tumors. CD82/KAI1 is known to associate with both integrins and RTKs. Our goal has been to determine how loss of CD82/KAI1 expression promotes metastasis by studying the role of CD82/KAI1 in integrin and RTK crosstalk.
We have previously shown that integrin activation of EGFR is required for integrinmediated induction of the Ras/Erk and PI3K/Akt signaling pathways. Recent studies indicate that integrin activation of Src depends on c-Met. Interestingly, inhibition of either Src or Ras/Erk signaling—but not PI3K/Akt signaling—induces cell death in primary prostate cells even when they are still adherent to matrix. Together these data indicate that Src signals generated by c-Met, as well 35
virtually untreatable. Metastasis and invasion by tumor cells require the activity of integrins, and in melanoma, expression of the αvβ3 integrin is induced during the development of invasive disease. Therefore, an understanding of how the αvβ3 integrin functions to regulate invasion will help our understanding of melanoma metastasis.
During prostate cancer progression there is a shift in the expression of laminin-specific integrins: β4 integrins are lost, and there is a concomitant increase in α6β1 and α3β1, which both interact with CD82/KAI1. We predict that the loss of CD82/KAI1 alters the function of α6β1 and α3β1. Using primary prostate epithelial cells, which express high levels of CD82/KAI1, as well as several prostate tumor cell lines that do not, we are exploring the role of CD82/KAI1 in regulating α6β1- and α3β1-mediated cell adhesion, migration, and integrin signaling. We have found that overexpression of CD82/KAI1 in tumor cells suppresses laminin-specific migration and invasion; integrin-induced EGFR and c-Met receptor activation; Src and Lyn activation; and activation of the Src/Lyn substrates Cas and FAK. Furthermore, integrin activation of Src is dependent on c-Met, and laminin-mediated invasion depends on both Src and c-Met. Together these data indicate that CD82/KAI1 normally acts to regulate integrin signaling to c-Met such that upon its loss of expression in tumor cells, signaling through c-Met to Src is increased, leading to increased motility and invasion (Fig. 2). We are currently determining the mechanism by which CD82/KAI1 down-regulates c-Met signaling. In reciprocal experiments, we are inhibiting the expression of CD82/KAI1 in primary cells using siRNA and mouse models.
The incidence of melanoma has been steadily increasing over the last 10 years. If diagnosed at an early stage, melanoma is curable, but once it has become invasive, it progresses very rapidly and is
We have been focusing our attention on two signaling molecules, PKCα and Src, both of which are regulated by the αvβ3 integrin and whose activities are enhanced in metastatic melanoma. Adhesion of normal melanocytes to extracellular matrix induces the formation of focal adhesion complexes and actin stress fibers. However, in a highly invasive metastatic melanoma cell line, C8161.9, these structures are absent. We have shown that the levels of Src activity and PKCα protein are elevated in these cells, and overexpression of PKCα in immortalized normal melanocytes is sufficient to confer an invasive phenotype in vitro. We have found that the activity of Rac, a small GTPase that is required for the formation of lamellipodia, is elevated in these cells and that inhibition of PKCα blocks Rac activity. The activity of the small GTPase Rho, which is involved in stress fiber and focal adhesion formation, is negatively regulated by active Src. Thus PKCα, acting to enhance Rac, and active Src, acting to inhibit Rho, together drive the formation of lamellipodia and the dissolution of stress fibers and focal adhesions, leading to increased motility (Fig. 3). We are currently exploring how Src is activated in melanoma cells, as well as the effects of blocking Src and PKCα expression with siRNA on migration and invasion.
Figure 2. CD82 reexpression in prostate tumor cells inhibits invasion by blocking integrinmediated signaling to Met and Src.
Figure 3. PKCa and Src cooperate to enhance migration and invasion in melanoma cells by differentially targeting Rac and Rho.
Integrin regulation of melanoma progression by PKC
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External Collaborators Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington Senthil Muthuswamy, Cold Spring Harbor Laboratory, New York Recent Publications Sridhar, S.C., and C.K. Miranti. In press. Tumor metastasis suppressor KAI1/CD82 is a tetraspanin. In Contemporary Cancer Research: Metastasis, C. Rinker-Schaeffer, M. Sokoloff, and D. Yamada, eds. Totowa, N.J.: Humana Press. Bill, Heather M., Beatrice Knudsen, Sheri L. Moores, Senthil K. Muthuswamy, Vikram R. Rao, Joan S. Brugge, and Cindy K. Miranti. 2004. Epidermal growth factor receptor–dependent regulation of integrin-mediated signaling and cell cycle entry in epithelial cells. Molecular and Cellular Biology 24(19): 8586–8599. Lee, Chong-Chou, Andrew J. Putnam, Cindy K. Miranti, Margaret Gustafson, Ling-Mei Wang, George F. Vande Woude, and Chong-Feng Gao. 2004. Overexpression of sprouty-2 inhibits HGF/SF-mediated cell growth, invasion, migration, and cytokinesis. Oncogene 23(30): 5193–5202.
From left to right: Schulz, Edick, Miranti, Long, Freiter, Sridhar
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Laboratory of Analytical, Cellular, and Molecular Microscopy and Laboratory of Microarray Technology and Molecular Diagnostics James H. Resau, Ph.D. Dr. Resau received his Ph.D. from the University of Maryland School of Medicine in 1985. He has been involved in clinical and basic science imaging and pathologyrelated research since 1972. Between 1968 and 1994, he was in the U.S. Army (active duty and reserve) and served in Vietnam. From 1985 until 1992, Dr. Resau was a tenured faculty member at the University of Maryland School of Medicine, Department of Pathology. Dr. Resau was the Director of the Analytical, Cellular and Molecular Microscopy Laboratory in the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute–Frederick Cancer Research and Development Center, Maryland, from 1992 to 1999. He joined VARI as a Special Program Senior Scientific Investigator in June 1999 and in 2003 was promoted to Deputy Director. In 2004, Dr. Resau assumed as well the direction of the Laboratory of Microarray Technology to consolidate the imaging and quantification of clinical samples in a CLIA-type research laboratory program. Laboratory Members
Staff Eric Kort, M.S. Bree Berghuis, B.S., HTL (ASCP), QIHC Pete Haak, B.S.
Consulting Veterinarian Robert Sigler, D.V.M., Ph.D.
Eric Hudson, B.S. Paul Norton, B.S. J.C. Goolsby
Students Hien Dang Brandon Leeser Amy Percival Huang Tran
Research Interests During 2004, we added two significant instruments to the ACMM laboratory. The first is the Aperio Scanscope, which enables us to digitize a 1 × 3-inch microscope slide in full color such that it can be analyzed, quantified, and shared throughout the digital network. The second is the Ventana automated immunostainer, which can carry out FISH, ISH, IHC, and array spotting in a programmed, specific, and accurate fashion. These two instruments have allowed us to increase our productivity, shorten our turnaround time, and improve the quality of our preparations without any increase in personnel. In the last calendar year, we processed 403 requests for histopathologic services that required 4,100 blocks and more than 31,600 glass slides. Using the Scanscope during December 2004, we scanned over 300 cases in high resolution for inclusion in the VATR database. We anticipate generating digital Scanscope files for 3,000–4,000 cases in 2005 that will be available on network servers. The MTMD laboratory is preparing gene expression data from cells and tissues and is correlating that with histology, tissue volume, and nuclear density to determine an effective and accurate
he Laboratory of Analytical, Cellular, and Molecular Microscopy (ACMM) and the Laboratory of Microarray Technology and Molecular Diagnostics (MTMD) are organized and equipped to produce highresolution images, genomic arrays, and bioinformatics data that support the cellular and molecular biology programs of the Institute. Our laboratories collaborate with the investigators to improve the understanding, diagnosis, and characterization of disease, injury, and differentiation. Although we primarily study cancer, we work with investigators in and out of the Institute on a variety of diseases and processes. As examples, during the past year, we have used our microscopes to produce intravital images of the Met protein in living cells and animals; localized and quantified a unique transcriptional protein in human cells; worked closely with VARI’s Bart Williams to characterize the phenotypic changes in transgenic mice expressing mutations related to Wnt and the Lrp5/Lrp6 genes; began to image Art Alberts’ small GTPase proteins in living cells; and collaborated in studies of the expression of c-Met in a series of primary human breast cancers.
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Original research within our labs focuses on quantification of images or arrays and the development of objective measurable data from images. A recent paper by Rozenblatt-Rosen et al. used a program written by Eric Kort for determining the co-localization of pixels that express unique fluorescent properties as well as the number of lumens or vessels in IHC-stained preparations. This is a direct extension of our Cytometry paper of 2003.
screen for applications in molecular diagnostic assays. We have developed a QC and QA protocol for evaluating specimens for array analysis. During the past year we have prepared 1,500 cDNA arrays for 20 collaborators from both in and outside the Institute. Our laboratories are primarily responsible for the archived clinical histopathology program called the Van Andel Tissue Repository (VATR). This program allows investigators to use existing human clinical samples to assess the expression of proteins. We also use these blocks to prepare a wide variety of tissue microarrays for research. We have increased the number of specimens in VATR to nearly 200,000 tissue samples. We are continuously entering data from the 200,000 blocks and now have 54,473 reports that further explain and describe the archives. The reports are not directly linked to any personal identifiers or names and meet all HIPAA/CLIA regulations. During this year we have begun the process of imaging representative blocks from the cases and adding demographic data. The material from future years will have digital information on age, sex, and diagnosis and will be linked to image files. These samples will be used in cellular and molecular protocols approved by our Institutional Review Board. Since its inception, the VATR program has been used by 24 registered users who have submitted 534 requests for searches and 101 subsequent tissue requests.
We have recently obtained funding to advance our understanding of breast cancer in collaboration with Ilan Tsarfaty, George Vande Woude, and Craig Webb. We have begun molecular imaging of breast cancer and correlation of the findings with Affymetrix gene expression. Other collaborations within VARI involve Met and HGF/SF in cells and tissues; the location of gene-targeted proteins in rodents; and the evaluation of monoclonal antibodies as diagnostic reagents. We have been funded by the Michigan Technology Tri-Corridor to develop the commercialization of diagnostic gene expression (collaboration with Bin Teh), imaging of primary tumors (Rick Hay), and expression of mRNA in human sperm as an indicator of fertility or sterility (Stephen Krawetz). We are in the early stages of developing a program in neuropathology with a concentration on Parkinson’s disease, starting with the morphological and genomic characterization of human adult stem-cell populations of specific neurons.
In collaboration with Rick Hay, we have augmented the VATR program with freshly frozen tissues from the tissue acquisition program. This HIPAA-compliant program involves the active cooperation of patients, surgeons, and pathologists from area hospitals, and the surgical tissues collected are used primarily in cDNA and Affymetrix gene expression studies. This collection also provides the participating physicians with access to research collaborations, with the aim of facilitating the translation of research results into clinical practice. The goal of this project is to develop genetics-based diagnostic classification of human disease. There is a Scientific Advisory Board for this project comprising members of VARI and of the Spectrum Health pathology, surgical and medical oncology, and surgery departments.
This year we have a student from Bath University in the U.K. The Bridges to the Baccalaureate program is a collaboration to support the recruitment of women and minorities into science careers; Dr. Resau is a coinvestigator and the VAI site coordinator for the program. In addition this year, we partnered with the Grand Rapids Area Pre-College Engineering Program (GRAPCEP) to build a school within a school for science education and instruction in Creston High School of the Grand Rapids Public School system. Our GRAPCEP mentorship program continues to be funded by Pfizer for a fifth year. Seven high school students have trained in the laboratory and are now in baccalaureate programs.
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External Collaborators Eric Arnoys and John Ubels, Calvin College, Grand Rapids, Michigan Stephan Baldus, University of Cologne, Germany Lonson Barr, Marcos Dantus, and Matti Koeppel, Michigan State University, East Lansing Nadia Harbeck, Ludwig-Maximilians-Universität, Munich, Germany Christine Hughes and O. Orit Rosen, Harvard University, Cambridge, Massachusetts Sylvia Kachalsky, Linkagene, Lod, Israel Iafa Keydar, Tel Aviv University, Israel Stephen Krawetz, Wayne State University, Detroit, Michigan Ernst Lengyel, University of Chicago, Illinois Maria Roberts, National Cancer Institute, Frederick, Maryland Rulong Shen, Ohio State University, Columbus Ilan Tsarfaty, Tel Aviv University, Israel Recent Publications Kort, E.J., M.R. Moore, E.A. Hudson, B. Leeser, G.M. Yeruhalmi, R. Leibowitz-Amit, G. Tsarfaty, I. Tsarfaty, S. Moshkovitz, and J.H. Resau. In press. Use of organ explant and cell culture. In Mechanisms of Carcinogenesis, Hans Kaiser, ed. Dordrecht, The Netherlands: Kluwer Academic. Lengyel, Ernst, Dieter Prechtel, James H. Resau, Katja Gauger, Anita Welk, Kristina Lindemann, Georgia Salanti, Thomas Richter, Beatrice Knudsen, George F. Vande Woude, and Nadia Harbeck. 2005. c-Met overexpression in node-positive breast cancer identifies patients with poor clinical outcome independent of Her2/neu. International Journal of Cancer 113(4): 678–682. Rozenblatt-Rosen, Orit, Christina M. Hughes, Suraj J. Nannepaga, Kalai Selvi Shanmugam, Terry D. Copeland, Tad Guszczynski, James H. Resau, and Matthew Meyerson. 2005. The parafibromin tumor suppressor protein is part of a human Paf1 complex. Molecular and Cellular Biology 25(2): 612–620. Seals, Darren F., Eduardo F. Azucena, Jr., Ian Pass, Lia Tesfay, Rebecca Gordon, Melissa Woodrow, James H. Resau, and Sara A. Courtneidge. 2005. The adaptor protein Tks5/Fish is required for podosome formation and function, and for the protease-driven invasion of cancer cells. Cancer Cell 7(2): 155–165. Akervall, Jan, Xiang Guo, Chao-Nan Qian, Jacqueline Schoumans, Brandon Leeser, Eric Kort, Andrew Cole, James Resau, Carol Bradford, Thomas Carey, Johan Wennerberg, Harald Anderson, Jan Tennvall, and Bin T. Teh. 2004. Genetic and expression profiles of squamous cell carcinoma of the head and neck correlate with cisplatin sensitivity and resistance in cell lines and patients. Clinical Cancer Research 10(24): 8204–8213. Birchenall-Roberts, Maria C., Tao Fu, Ok-sun Bang, Michael Dambach, James H. Resau, Cari L. Sadowski, Daniel C. Bertolette, Ho-Jae Lee, Seong-Jin Kim, and Francis W. Ruscetti. 2004. Tuberous sclerosis complex 2 gene product interacts with human SMAD proteins. Journal of Biological Chemistry 279(24): 25605–25613. Holmen, Sheri L., Troy A. Giambernardi, Cassandra R. Zylstra, Bree D. Buckner-Berghuis, James H. Resau, J. Fred Hess, Vaida Glatt, Mary L. Bouxsein, Minrong Ai, Matthew L. Warman, and Bart O. Williams. 2004. Decreased BMD and limb deformities in mice carrying mutations in both Lrp5 and Lrp6. Journal of Bone and Mineral Research 19(12): 2033–2040. Tan, Min-Han, Carl Morrison, Pengfei Wang, Ximing Yang, Carola J. Haven, Chun Zhang, Ping Zhao, Maria S. Tretiakova, Eeva Korpi-Hyovalti, John R. Burgess, Khee Chee Soo, Wei-Keat Cheah, Brian Cao, James Resau, Hans Morreau, and Bin Tean Teh. 2004. Loss of parafibromin immunoreactivity is a distinguishing feature of parathyroid carcinoma. Clinical Cancer Research 10(19): 6629–663 40
From left to right: Resau, Goolsby, Haak, Berghuis, Norton, Percival, Hudson, Ferrell
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Laboratory of Germline Modification Pamela J. Swiatek, Ph.D., M.B.A. Dr. Swiatek received her M.S. (1984) and Ph.D. (1988) degrees in pathology from Indiana University. From 1988 to 1990, she was a postdoctoral fellow at the Tampa Bay Research Institute. From 1990 to 1994, she was a postdoctoral fellow at the Roche Institute of Molecular Biology in the laboratory of Tom Gridley. From 1994 to 2000, Dr. Swiatek was a Research Scientist and Director of the Transgenic Core Facility at the Wadsworth Center in Albany, N.Y., and an Assistant Professor in the Department of Biomedical Sciences at the State University of New York at Albany. She joined VARI as a Special Program Investigator in August 2000. She has been the chair of the Institutional Animal Care and Use Committee since 2002 and is an Adjunct Assistant Professor in the College of Veterinary Medicine at Michigan State University. Dr. Swiatek received her M.B.A. in 2005 from Krannert School of Management at Purdue University. Laboratory Members
Staff Julie Koeman, B.S. Kellie Sisson, B.S. Juraj Zahatnansky, B.S.
IACUC Coordinator Kaye Johnson, B.S.
Research Interests missing, cause the mouse to die as an embryo. The lab also has the capability to produce mutant embryos that have a wild-type placenta using tetraploid embryo technology. This technique is useful when the gene-targeted mutation prevents implantation of the mouse embryo in the uterus. We also assist in the development of embryonic stem (ES) or fibroblast cell lines from mutant embryos, which allows for in vitro studies of the gene mutation.
T
he germline modification lab is a fullservice lab that functions at the levels of service, research, and teaching to develop, analyze, and maintain mouse models of human disease. Our lab applies a business philosophy to core service offerings and we focus on scientific innovation, customer satisfaction, and service excellence. Mouse models are produced using gene-targeting technology, a wellestablished, powerful method for inserting specific genetic changes into the mouse genome. The resulting mice can be used to study the effects of these changes in the complex biological environment of a living organism. The genetic changes can include the introduction of a gene into a specific site in the genome, (gene “knockin”) or the inactivation of a gene already in the genome (gene “knock-out”). Since these mutations are introduced into the reproductive cells known as the germline, they can be used to study the developmental aspects of gene function associated with inherited genetic diseases.
Our gene-targeting service encompasses three major procedures: DNA electroporation, clone expansion and cryopreservation, and microinjection. Gene targeting procedures are initiated by mutating the genomic DNA of interest and inserting it into ES cells using the electroporation technique. The mutated gene integrates into the genome of the ES cells and, by a process called homologous recombination, replaces one of the two wild-type copies of the gene in the cells. Clones are identified, isolated, and cryopreserved, and genomic DNA is extracted from each clone and delivered to the client for analysis. Correctly targeted ES cell clones are thawed, established into tissue culture, and cryopreserved in liquid nitrogen. Gene-targeting mutations are introduced into the mouse by microinjection of the pluripotent ES cell clones into 3.5-day-old mouse embryos (blastocysts). These embryos, containing a mixture of wild-type
In addition to traditional gene-targeting technologies, the germline modification lab can produce mouse models in which the gene of interest is inactivated in a target organ or cell line instead of in the entire animal. These types of mouse models, known as conditional knock-outs, are particularly useful in studying genes that, if 42
cost-effective procedures decrease the need to continuously breed valuable mouse models, and they provide added insurance against the loss of custom mouse lines due to disease outbreak or a catastrophic event. Eight-cell mouse embryos or mouse sperm can be cryopreserved and stored in liquid nitrogen; they can be reconstituted by implantation into the oviducts of recipient mice or by in vitro fertilization of oocytes, respectively.
and mutant ES cells, develop into mice called chimeras. The offspring of chimeras that inherit the mutated gene are heterozygotes, because they possess one copy of the mutated gene. The heterozygous mice are bred together to produce mice that completely lack the normal gene. These homozygous mice have two copies of the mutant gene and are called knock-out mice. Once gene targeting mice are produced, our lab assists in developing breeding schemes and provides for complete analysis of the mutants. The efficiency of mutant mouse production and analysis is enhanced by the AutoGenprep 960, a robotic, high-throughput DNA isolation machine. Tail biopsies from genetically engineered mice are processed in a 96-well format and the DNA samples are delivered to the client for analysis. It is our future plan that the DNA analysis be fully automated, with samples moving directly from the AutoGenprep 960 to a high-throughput genotyping platform, eliminating the need for clients to perform this labor-intensive analysis.
The VARI germline modification lab directs the Michigan Animal Model Consortium (MAMC) of the Core Technology Alliance (CTA) Corp. MAMC is one of six collaborative core facilities located at the University of Michigan, Michigan State University, Wayne State University, Kalamazoo Community College, and VARI, offering research services in proteomics, structural biology, genomics, bioinformatics, high-throughput compound screening, and animal modeling. These labs receive funding from the Michigan Economic Development Corporation to efficiently provide mouse modeling services to researchers studying human diseases and to promote the commercialization of the core services in order to stimulate the development of biomedical research in Michigan.
The germline modification lab also directs the VARI cytogenetics core, which offers a variety of custom services. Mouse, rat, and human cell lines derived from tumors, fibroblasts, blood, or ES cells can be grown in tissue culture, growtharrested, fixed, and spread onto glass slides. Karyotyping of chromosomes using Leishman- or Giemsa-stained (G-banded) chromosomes is our basic service. However, spectral karyotyping (SKY) analysis of metaphase chromosome spreads, using high-quality, 24-color, wholechromosome fluorescent paints, can aid in the detection of subtle and complex chromosomal rearrangements. Fluorescence in situ hybridization (FISH) analysis, using indirectly or directly labeled bacterial artificial chromosome (BAC) or plasmid probes, can also be performed on metaphase spreads or on interphase nuclei derived from tissue touch preps or nondividing cells. Sequential staining of identical metaphase spreads using FISH and SKY can assist in identifying the chromosome integration site of a randomly integrated transgene.
The MAMC services are classified into three major categories: mouse model development; analysis; and maintenance and preservation. Model development services consist of gene targeting, transgenic, TVA transgenic, and xenotransplantation procedures. Analytical procedures are performed on the animal models to determine the nature and extent of any phenotypic consequences in the models and their correlation with human disease. Mouse analysis consists of histology, necropsy, veterinary pathology, cytogenetics, blood chemistry, blood hematology, and imaging. The DNA isolation service supports the genotyping analysis of the models, and the monoclonal antibody core supports the molecular analysis of the mutant mice and xenotransplantation models. Finally, the maintenance and preservation services include a mouse repository, mouse breeding services, mouse rederivation, and embryo/sperm cryopreservation. These services are described more completely on the MAMC website at <www.vai.org/core/mouse>.
Finally the germline modification lab provides cryopreservation services for archiving and reconstituting valuable mouse strains. These
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Recent Publications Robertson, Scott A., Jacqueline Schoumans, Brendan D. Looyenga, Jason A. Yuhas, Cassandra R. Zylstra, Julie M. Koeman, Pamela J. Swiatek, Bin T. Teh, and Bart O. Williams. 2005. Spectral karyotyping of sarcomas and fibroblasts derived from Ink4a/Arf-deficient mice reveals chromosomal instability in vitro. International Journal of Oncology 26(3): 629–634. Wu, Lin, Jun Gu, Huadong Cui, Qing-Yu Zhang, Melissa Behr, Cheng Fang, Yan Weng, Kerri Kluetzman, Pamela J. Swiatek, Weizhu Yang, Laurence Kaminsky, and Xinxin Ding. 2005. Transgenic mice with a hypomorphic NADPH-cytochrome P450 reductase gene: effects on development, reproduction, and microsomal cytochrome P450. Journal of Pharmacology and Experimental Therapeutics 312(1): 35–43. Graveel, Carrie, Yanli Su, Julie Koeman, Ling-Mei Wang, Lino Tessarollo, Michelle Fiscella, Carmen Birchmeier, Pamela Swiatek, Roderick Bronson, and George Vande Woude. 2004. Activating Met mutations produce unique tumor profiles in mice with selective duplication of the mutant allele. Proceedings of the National Academy of Sciences U.S.A. 101(49): 17198–17203.
From left to right: Swiatck, Koeman, Zahatnansky, Sisson
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Laboratory of Cancer Genetics Bin T. Teh, M.D., Ph.D. Dr. Teh obtained his M.D. from the University of Queensland, Australia, in 1992, and his Ph.D. from the Karolinska Institute, Sweden, in 1997. Before joining the Van Andel Research Institute (VARI), he was an Associate Professor of medical genetics at the Karolinska Institute. Dr. Teh joined VARI as a Senior Scientific Investigator in January 2000. Dr. Teh’s research mainly focuses on kidney cancer, and he is currently on the Medical Advisory Board of the Kidney Cancer Association. He became VARI’s Deputy Director for Research Operations in the fall of 2003 and was promoted to Distinguished Scientific Investigator in 2005. Laboratory Members
Staff Miles Chao-Nan Qian, M.D., Ph.D. Pengfei Wang, M.D., Ph.D. Xin Yao, M.D., Ph.D. Jindong Chen, Ph.D. Kunihiko Futami, Ph.D.
Sok Kean Khoo, Ph.D. Douglas Luccio-Camelo, Ph.D. David Petillo, Ph.D. Eric Kort, M.S. Stephanie Potter, M.S.
Jeff Bates, B.S. Timothy Yaw Bediako, B.S. Mark Betten, B.S. Aaron Massie, B.S.
Research Interests (NORE1 and LSAMP) by us and others, we are now focusing on functional studies of these genes, including generating mouse models that harbor mutations of these genes (see below). We established the role of HRPT2 in parathyroid carcinoma by mutation analysis and immunohistochemical staining. We have shown that its protein, parafibromin, accumulates predominantly in the nucleus, and this nuclear localization is essential to maintaining its antiproliferative function.
K
idney cancer, or renal cell carcinoma (RCC), is the tenth most common cancer in the United States (34,000 new cases and more than 12,000 deaths a year). Its incidence has been increasing, a phenomenon that cannot be accounted for by the wider use of imaging procedures. We have established a comprehensive and integrated kidney research program, and our major research goals are 1) to identify the molecular signatures of different subtypes of kidney tumors, both hereditary and sporadic, and to understand how these genes function and interact in giving rise to the tumors and their progression; 2) to identify and develop novel biomarkers and key drug targets; and 3) to generate animal models for drug testing and preclinical bioimaging.
Microarray gene expression profiling and bioinformatics Based on microarray profiling of 400 kidney tumors using both our own spotted arrays and Affymetrix microarrays, we have identified the molecular signatures for 1) different subtypes of kidney tumors; 2) the prognosis for clear cell RCC and papillary RCC; and 3) the prediction of drug response (immunotherapy). Our studies have been validated by RT-PCR and immunohistochemical staining. We have also been working closely with VARI’s Kyle Furge in analyzing our microarray data. Using a program developed by his team, comparative genomic microarray analysis (CGMA), we correlated gene expression profiles with the predicted chromosomal imbalances for different RCC histological subtypes, which may facilitate our efforts in identifying RCC-related genes from these chromosomal regions. More information and details can be found in several of our review articles and book chapters.
Our program to date has established a worldwide network of collaborators, a tissue bank containing fresh-frozen tumor pairs (over 700 cases) and serum, and a gene expression profiling database of 500 tumors with long-term clinical follow-up information for half of them. Our program includes positional cloning of hereditary RCC syndromes and functional studies of their related genes, microarray and bioinformatic analysis, and generation of RCC mouse models. Hereditary RCC syndromes – positional cloning and functional studies Following the identification of the HRPT2, BHD, and two familial RCC breakpoint genes 45
Mouse models of kidney cancer
epithelial-specific Cre/lox recombination in these tissues. We then crossed these mice with the APC-, VHL-, and PTEN-floxed mice. To date, we have completed the studies on APC conditional knock-outs, which give the phenotype of polycystic kidney disease and renal adenoma. We are currently characterizing the phenotypes of the other mice. In addition, we have established xenograft subcapsular models using five different RCC cell lines. The natural course of these models has been documented and studied by microarrary gene expression profiling.
In collaboration with VARI’s Bart Williams and Pam Swiatek, we have set up several mouse models for kidney tumors. These include conventional and conditional knock-outs for BHD, HRPT2, VHL, PTEN, and APC. For the latter three cases, we collaborated with Peter Igarashi of the University of Texas Southwestern Medical Center and obtained from him Ksp1.3/Cre transgenic mice expressing Cre recombinase exclusively in the kidney and developing GU tract, which can mediate
External Collaborators We have extensive collaborations with researchers and clinicians in the United States and overseas. Recent Publications Morris, M.R., D. Gentle, M. Abdulrahman, E.N. Maina, K. Gupta, R.E. Banks, M.S. Wiesener, T. Kishida, M. Yao, B. Teh, F. Latif, and E.R. Maher. In press. Tumor suppressor activity and epigenetic inactivation of hepatocyte growth factor activator inhibitor type 2 (HAI-2/SPINT2) in papillary and clear cell renal cell carcinoma. Cancer Research. Takahashi, M., X.J. Yang, S. McWhinney, N. Sano, C.H. Eng, S. Kagawa, B.T. Teh, and H. Kanayama. In press. cDNA microarray analysis assists in diagnosis of malignant intrarenal pheochromocytoma originally masquerading as a renal cell carcinoma. Journal of Medical Genetics. Tretiakova, M., M. Turkyilmaz, T. Grushko, M. Kocherginsky, C. Rubin, O. Olopade, B.T. Teh, and X.J. Yang. In press. Topoisomerase II[α] expression in Wilms tumors. Clinical Cancer Research. Wang, P., M.-H. Tan, C. Zhang, H. Morreau, and B.T. Teh. In press. HRPT2, a tumor suppressor gene for hyperparathyroidism-jaw tumor syndrome. Hormone and Metabolic Research. Yang, X.J., M.-H. Tan, H.L. Kim, J.A. Ditlev, M.W. Betten, C.E. Png, E.J. Kort, K. Futami, K.J. Dykema, K.A. Furge, M. Takahashi, H. Kanayama, P.H. Tan, B.S. Teh, C. Luan, et al. In press. A molecular classification of papillary renal cell carcinoma. Cancer Research. Cardinal, J., L. Bergman, N. Hayward, A. Sweet, J. Warner, L. Marks, D. Learoyd, T. Dwight, B. Robinson, M. Epstein, M. Smith, B.T. Teh, D. Cameron, and J. Prins. 2005. A report of a national mutation testing service for the MEN1 gene: clinical presentations and implications for mutation testing. Journal of Medical Genetics 42(1): 69–74. Chuang, S.T., P. Chu, J. Sugimura, M.S. Tretiakova, V. Papavero, K. Wang, M.-H. Tan, F. Lin, B.T. Teh, and X.J. Yang. 2005. Overexpression of glutathione S-transferase α in clear cell renal cell carcinoma. American Journal of Clinical Pathology 123(3): 421–429. Lee, Youn-Soo, Alexander O. Vortmeyer, Irina A. Lubensky, Timothy W.A. Vogel, Barbara Ikejiri, Sophie Ferlicot, Gérard Benoît, Sophie Giraud, Edward H. Oldfield, W. Marston Linehan, Bin T. Teh, Stéphane Richard, and Zhengping Zhuang. 2005. Coexpression of erythropoietin and erythropoietin receptor in Von Hippel-Lindau disease–associated renal cysts and renal cell carcinoma. Clinical Cancer Research 11(3): 1059–1064. Qian, Chao-Nan, Jared Knol, Peter Igarashi, Fangmin Lin, Uko Zylstra, Bin Tean Teh, and Bart O. Williams. 2005. Cystic renal neoplasia following conditional inactivation of Apc in mouse renal tubular epithelium. Journal of Biological Chemistry 280(5): 3938–3945. Robertson, Scott A., Jacqueline Schoumans, Brendan D. Looyenga, Jason A. Yuhas, Cassandra R. Zylstra, Julie M. Koeman, Pamela J. Swiatek, Bin T. Teh, and Bart O. Williams. 2005. Spectral 46
karyotyping of sarcomas and fibroblasts derived from Ink4a/Arf-deficient mice reveals chromosomal instability in vitro. International Journal of Oncology 26(3): 629–634. Rogers, C., M.-H. Tan, and B.T. Teh. 2005. Gene expression profiling of renal cell carcinoma and clinical implications. Urology 65(2): 231–237. Schoumans, Jacqueline, Ann Nordgren, Claudia Ruivenkamp, Karen Brøndum-Nielsen, Bin Tean Teh, Göran Annéren, Eva Holmberg, Magnus Nordenskjöld, and Britt-Marie Anderlid. 2005. Genome-wide screening using array-CGH does not reveal microdeletions/microduplications in children with Kabuki syndrome. European Journal of Human Genetics 13(2): 260–263. Takahashi, Masayuki, Veronica Papavero, Jason Yuhas, Eric Kort, Hiro-omi Kanayama, Susumu Kagawa, R.C. Baxter, Ximing J. Yang, Steven G. Gray, and Bin T. Teh. 2005. Altered expression of members of the IGF-axis in clear cell renal cell carcinoma. International Journal of Oncology 26(4): 923–931. Akervall, Jan, Xiang Guo, Chao-Nan Qian, Jacqueline Schoumans, Brandon Leeser, Eric Kort, Andrew Cole, James Resau, Carol Bradford, Thomas Carey, Johan Wennerberg, Harald Anderson, Jan Tennvall, and Bin T. Teh. 2004. Genetic and expression profiles of squamous cell carcinoma of the head and neck correlate with cisplatin sensitivity and resistance in cell lines and patients. Clinical Cancer Research 10(24): 8204–8213. Atkins, Michael B., David E. Avigan, Ronald M. Bukowski, Richard W. Childs, Janice P. Dutcher, Tim G. Eisen, Robert A. Figlin, James H. Finke, Robert C. Flanigan, Daniel J. George, S. Nahum Goldberg, Michael S. Gordon, Othon Iliopoulos, William G. Kaelin, Jr., W. Marston Linehan, et al. 2004. Innovations and challenges in renal cancer: consensus statement. Clinical Cancer Research 10(18, Pt.2): 6277S–6281S. Calender, A., C. Morrison, P. Komminoth, J.Y. Scaoazec, K. Sweet, and B.T. Teh. 2004. Multiple endocrine neoplasia type 1. In Pathology and Genetics of Tumours of the Endocrine Organs, DeLellis, Heitz, Lloyd, and Eng, eds. WHO Classification of Tumours series, Vol. 8. Lyon, France: IARC Press, pp. 218–227. Furge, Kyle A., Kerry A. Lucas, Masayuki Takahashi, Jun Sugimura, Eric J. Kort, Hiro-omi Kanayama, Susumu Kagawa, Philip Hoekstra, John Curry, Ximing J. Yang, and Bin T. Teh. 2004. Robust classification of renal cell carcinoma based on gene expression data and predicted cytogenetic profiles. Cancer Research 64(12): 4117–4121. Haven, Carola J., Viive M. Howell, Paul H.C. Eilers, Robert Dunne, Masayuki Takahashi, Marjo van Puijenbroek, Kyle Furge, Job Kievit, Min-Han Tan, Gert Jan Fleuren, Bruce G. Robinson, Leigh W. Delbridge, Jeanette Philips, Anne E. Nelson, Ulf Krause, et al. 2004. Gene expression of parathyroid tumors: molecular subclassification and identification of the potential malignant phenotype. Cancer Research 64(20): 7405–7411. Lindvall, Charlotta, Kyle Furge, Magnus Björkholm, Xiang Guo, Brian Haab, Elisabeth Blennow, Magnus Nordenskjöld, and Bin Tean Teh. 2004. Combined genetic and transcriptional profiling of acute myeloid leukemia with normal and complex karyotypes. Haematologica 89(9): 1072–1081. Luccio-Camelo, Douglas C., Karina N. Une, Rafael E.S. Ferreira, Sok Kean Khoo, Radoslav Nickolov, Marcello D. Bronstein, Mario Vaisman, Bin Tean Teh, Lawrence A. Frohman, Berenice B. Mendonca, and Monica R. Gadelha. 2004. A meiotic recombination in a new isolated familial somatotropinoma kindred. European Journal of Endocrinology 150(5): 643–648. Marsh, Deborah J., Hans Morreau, and Bin T. Teh. 2004. HRPT2 and parathyroid cancer. Lancet Oncology 5(2): 78. Morrison, C., K. Sweet, and B.T. Teh. 2004. Hyperthyroidism-jaw tumor syndrome. In Pathology and Genetics of Tumours of the Endocrine Organs, DeLellis, Heitz, Lloyd, and Eng, eds. WHO Classification of Tumours series, Vol. 8. Lyon, France: IARC Press, pp. 228–237. Patton, Kurt T., Maria S. Tretiakova, Jorge L. Yao, Veronica Papavero, Lei Huo, Brian P. Adley, Guan Wu, Jiaoti Huang, Michael R. Pins, Bin T. Teh, and Ximing J. Yang. 2004. Expression of RON proto-oncogene in renal oncocytoma and chromophobe renal cell carcinoma. American Journal of Surgical Pathology 28(8): 1045–1050. 47
Stephens, P., C. Hunter, G. Bignell, S. Edkins, H. Davies, J. Teague, C. Stevens, S. O’Meara, R. Smith, A. Parker, A. Barthorpe, M. Blow, L. Brackenbury, A. Butler, O. Clarke, et al. 2004. Lung cancer: intragenic ERBB2 kinase mutations in tumours. Nature 431(7008): 525–526. Sugimura, Jun, Richard S. Foster, Oscar W. Cummings, Eric J. Kort, Masayuki Takahashi, Todd T. Lavery, Kyle A. Furge, Lawrence H. Einhorn, and Bin Tean Teh. 2004. Gene expression profiling of early- and late-relapse nonseminomatous germ cell tumor and primitive neuroectodermal tumor of the testis. Clinical Cancer Research 10(7): 2368–2378. Sugimura, Jun, Ximing J. Yang, Maria S. Tretiakova, Masayuki Takahashi, Eric J. Kort, Barbara Fulton, Tomoaki Fujioka, Nicholas J. Vogelzang, and Bin Tean Teh. 2004. Gene expression profiling of mesoblastic nephroma and Wilms tumors—comparison and clinical implications. Urology 64(2): 362–368. Tan, Min-Han, Carl Morrison, Pengfei Wang, Ximing Yang, Carola J. Haven, Chun Zhang, Ping Zhao, Maria S. Tretiakova, Eeva Korpi-Hyovalti, John R. Burgess, Khee Chee Soo, Wei-Keat Cheah, Brian Cao, James Resau, Hans Morreau, and Bin Tean Teh. 2004. Loss of parafibromin immunoreactivity is a distinguishing feature of parathyroid carcinoma. Clinical Cancer Research 10(19): 6629–6637. Tan, Min-Han, Craig G. Rogers, Jeffrey T. Cooper, Jonathon A. Ditlev, Thomas J. Maatman, Ximing Yang, Kyle A. Furge, and Bin Tean Teh. 2004. Gene expression profiling of renal cell carcinoma. Clinical Cancer Research 10(18): 6315S–6321S. Tan, M.-H., and B.T. Teh. 2004. Renal neoplasia in the hyperparathyroidism-jaw tumor syndrome. Current Molecular Medicine 4(8): 895–897. Teh, B.T. 2004. Gene expression profiling begins to fulfill promise in differential diagnosis and prognosis of renal cell carcinomas. Kidney Cancer Journal 2(3): 15–18.
Standing, left to right: Qian, Wang, Massie, Petillo, Bates, Bediako, Chen seated, left to right: Betten, Khoo, Antio, Potter, Teh, Futami
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Laboratory of Molecular Oncology George F. Vande Woude, Ph.D. Dr. Vande Woude received his M.S. (1962) and Ph.D. (1964) from Rutgers University. From 1964â&#x20AC;&#x201C;1972, he served first as a postdoctoral research associate, then as a research virologist for the U.S. Department of Agriculture at Plum Island Animal Disease Center. In 1972, he joined the National Cancer Institute as Head of the Human Tumor Studies and Virus Tumor Biochemistry sections and, in 1980, was appointed Chief of the Laboratory of Molecular Oncology. In 1983, he became Director of the Advanced Bioscience Laboratoriesâ&#x20AC;&#x201C;Basic Research Program at the National Cancer Instituteâ&#x20AC;&#x2122;s Frederick Cancer Research and Development Center, a position he held until 1998. From 1995, Dr. Vande Woude first served as Special Advisor to the Director, and then as Director for the Division of Basic Sciences at the National Cancer Institute. In 1999, he was recruited to the Directorship of the Van Andel Research Institute in Grand Rapids, Michigan. Laboratory Members
Staff George Vande Woude, Ph.D. Rick Hay, Ph.D., M.D. Yu-Wen Zhang, M.D., Ph.D. Chongfeng Gao, Ph.D. Carrie Graveel, Ph.D.
Sharon Moshkovitz, Ph.D. Qian Xie, Ph.D. Dafna Kaufman, M.Sc. Lia Tesfay, M.S. Matt VanBrocklin, M.S.
Benjamin Staal, B.S. Yanli Su, A.M.A.T.
Visiting Scientists Galia Tsarfaty, M.D. Ilan Tsarfaty, Ph.D. Student Jack DeGroot
Research Interests Proliferation and invasion
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ctivating mutations in Met are found in human kidney and gastric cancers, providing compelling genetic evidence that Met is an important oncogene (http://www.vai.org/vari/metandcancer/). To study how activating mutations are involved in tumor development, we generated mice bearing Met with mutations representing both inherited and sporadic mutations found in human cancers. The different mutant Met lines developed unique tumor profiles including carcinomas, sarcomas, and lymphomas. Cytogenetic analysis of the tumors in Met mutant mice shows that in all cases amplification of the mutant met allele is observed. Selective chromosomal amplification is also found in patients with renal cancer, indicating that amplification of the mutant met allele may be required for tumor progression.
We are interested in how HGF/SF-induced proliferation and invasion contribute to tumor progression. We have established in vitro methods to select highly proliferative or invasive cell populations that may mimic the in vivo process of clonal selection during tumor progression. Our studies show that most tumor cells display both invasive and proliferative phenotypes and that they can reversibly change from invasive to proliferative phenotypes. We are currently studying the genetic and epigenetic factors that determine the different cellular responses. HGF/SF-Met-mediated tumor angiogenesis HGF/SF acts as an angiogenic switch by simultaneously up-regulating vascular endothelial growth factor (VEGF) and down-regulating thrombospondin 1 (TSP-1) expression in the same tumor cells, mediated through the MAPK pathway. In addition, HGF/SF also downregulates TSP-1 expression in normal human umbilical vascular endothelial cells, in which VEGF expression is undetectable. TSP-1 and inhibitors of VEGF (such as Avastin) have been shown to inhibit tumor angiogenesis and growth. Our data raise the question of whether TSP-1 in combination with VEGF inhibitors would enhance inhibition of HGF/SF-Met-mediated
The differences in tumor types and latency, depending on the mutation, may be due to signaling differences triggered by the specific mutation. Studies are underway to identify the signaling pathways selectively activated by these mutations. Understanding the signaling specificity of these mutations is essential to identifying and developing successful therapeutics. Our mutant mice provide a valuable model for testing Met inhibitors and for understanding the molecular events critical for Met-mediated tumorigenesis. 49
Imaging of Met oncogene activation
tumor angiogenesis and growth relative to each tested separately, and whether these inhibitors are better than those specifically directed against HGF/SF or Met receptor. We are testing these approaches.
We have generated mice carrying a murine GFP-Met transgene that emits intense fluorescence to reveal where in the animal Met is expressed. Five founder mice were selected that had GFP-Met expression levels from high to moderate. All of the transgenic GFP-Met male miceâ&#x20AC;&#x201D;but no femalesâ&#x20AC;&#x201D;develop tumors in the preputial sebaceous glands. Moreover, mice expressing the highest levels of the GFP-Met transgene develop tumors earlier. However, tumors originating from the different founder lines had similar pathological phenotypes; the mice presented with cystic sebaceous tumors having a rapid growth rate. GFP-Met expression is always higher in the sebaceous gland tumors relative to normal skin, with tumors covering the spectrum of adenomas, adenocarcinomas, and angiosarcomas. Metastases developed in 71% of GFP-Met transgenic mice with adenocarcinoma and in 18% of mice with angiosarcoma. The metastases were found locally in the skin and in distant organs such as liver, lung, and kidney. Image analyses of unfixed frozen tumor metastases showed large numbers of cells overexpressing GFP-Met, and tissue arrays of these tumors revealed higher GFP-Met levels relative to the primary tumors.
Immunocompromised transgenic mice with Met-expressing xenografts We have generated a severe combined immune deficiency (SCID) mouse strain carrying a human HGF/SF transgene. This mouse provides a species-compatible ligand for propagating human tumor cells expressing human Met receptors. The growth of Met-expressing human tumor xenografts can be significantly enhanced in this transgenic mouse relative to those in nontransgenic hosts. Our data strongly suggest that this immunocompromised strain will be a useful tool for investigating the role of Met in tumor malignancy. Currently, we are testing experimental metastases and orthotopic xenografts of human tumor cells. This model will also be used for preclinical testing of drugs or compounds targeting the HGF/SF-Met complex and downstream signaling pathways. Geldanamycin inhibits tumor cell invasion at femtomolar concentrations Our lab has been studying the mechanism of geldanamycin (GA) inhibition of urokinase activation of plasmin from plasminogen (uPA). Previously, we have shown that a subset of GA derivatives at femtomolar concentrations (fMGAi) inhibit HGF/SF-induced activation of plasmin in canine MDCK cells. We have recently found that such inhibition also occurs in several human glioblastoma cell lines (DBTRG, U373, and SNB19) and in SK-LMS-1 human leiomyosarcoma cells. Curiously, fM-GAi drugs only inhibit HGF/SF-induced uPA activity, and only when the magnitude of HGF/SF-uPA induction is above 1.5 times basal uPA activity. These fM-GAi derivatives also block MDCK cell scattering and glioblastoma tumor cell invasion in vitro at concentrations well below those required to exhibit a measurable effect on Met expression. Other experiments using radicicol and macbecin II provide circumstantial evidence for a novel non-HSP90 molecular target that is involved in HGF/SF-mediated tumor cell invasion.
MAPK in melanoma Constitutive activation of MAPK signaling contributes to many human cancers, including melanoma, with activating mutations in Nras or BRAF found in 80% of the tumors. While most cancer cells exhibit a cytostatic response to disruption of MAPK signaling, melanomas show a cytotoxic response. The Koo laboratory showed that inhibition of MAPK signaling efficiently triggers cell death by apoptosis in human melanoma cells. Normal epidermal melanocytes, however, do not undergo apoptosis in response to MAPK inhibition, but arrest in the G1 phase of the cell cycle. Moreover, in vivo interference of MAPK signaling produces either significant or complete regression of human melanoma xenograft tumors in athymic nude mice. These results indicate that the MAPK pathway represents tumor-specific survival signaling in melanoma, and that inhibition of this pathway may be a therapeutic strategy. 50
Nuclear oncology
diagnostic tools in this setting. Preliminary experiments (performed in collaboration with David Wenkert and Milton Gross) have shown that radiometal chelation (e.g., with Tc-99m pertechnetate) may be used instead of direct radioiodination for labeling MetSeek mAbs. In collaboration with David Waters, we have conducted a short-term analysis of nonradioactive Met5 in normal dogs. No clinical, hematological, or chemical evidence of toxicity has been found, and histopathological analysis of tissues harvested at necropsy is pending.
During the past year our laboratory has made progress in the area of nuclear oncology on two fronts: the continued development of full-length anti-Met monoclonal antibodies (mAbs) for radioimmunodiagnostic and therapeutic applications and the new development of genetically engineered Met-directed antibody fragments. We call our biological agents that recognize human Met “MetSeek” and we continue to evaluate two full-length mAbs, Met3 and Met5, in animal models of cancer. Met3 recognizes an epitope on the extracellular domain of human Met, but not on canine Met, whereas Met5 recognizes an epitope common to human and canine Met. Experiments on the usefulness of radiolabeled Met3 for imaging human glioblastoma multiforme tumor xenografts (because of their exquisite sensitivity to geldanamycins) are in progress. We have a new collaboration with David Schteingart to study Met expression by human adrenocortical carcinomas and to evaluate MetSeek mAbs as
We have launched a collaborative project to develop and evaluate genetically engineered Met-directed antibody fragments as alternatives to full-length MetSeek mAbs for diagnostic and therapeutic applications. Single-chain versions of Met3 and Met5 (scFv) are under construction at ApoLife, Inc., a biotechnology firm in Detroit, and we have shown that a new Met-directed human-Fab-expressing phage display product can be used to image autocrine human leiomyosarcoma (SK-LMS-1/HGF) tumor xenografts in nude mice.
External Collaborators Milton Gross, Department of Veterans Affairs Healthcare System, Ann Arbor, Michigan Nadia Harbeck, Technische Universität, Munich, Germany Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington Ernest Lengyel, University of Chicago, Illinois Alnawaz Rehemtulla, Brian Ross, and David Schteingart, University of Michigan, Ann Arbor Yuehai Shen and David Wenkert, Michigan State University, East Lansing Olga Volpert, Northwestern University, Evanston, Illinois David Waters, Gerald P. Murphy Cancer Foundation, West Lafayette, Indiana Robert Wondergem, East Tennessee State University, Johnson City Recent Publications Jiao, Y., P. Zhao, J. Zhu, T. Grabinski, Z. Feng, X. Guan, R.S. Skinner, M.D. Gross, Y. Su, G.F. Vande Woude, R.V. Hay, and B. Cao. In press. Construction of human naïve Fab library and characterization of anti-Met Fab fragment generated from the library. Molecular Biotechnology. Gao, Chong Feng, and George F. Vande Woude. 2005. HGF/SF signaling in tumor progression. Cell Research 15(1): 49–51. Islam, Azharul, Yoko Sakamoto, Kazuhisa Kosaka, Satoshi Yoshitome, Isamu Sugimoto, Kazuo Yamada, Ellen Shibuya, George F. Vande Woude, and Eikichi Hashimoto. 2005. The distinct stage-specific effects of 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid on the activation of MAP kinase and Cdc2 kinase in Xenopus oocyte maturation. Cellular Signalling 17(4): 507–523. Lengyel, Ernst, Dieter Prechtel, James H. Resau, Katja Gauger, Anita Welk, Kristina Lindemann, Georgia Salanti, Thomas Richter, Beatrice Knudsen, George F. Vande Woude, and Nadia Harbeck. 51
2005. c-Met overexpression in node-positive breast cancer identifies patients with poor clinical outcome independent of Her2/neu. International Journal of Cancer 113(4): 678–682. Zhang, Yu-Wen, Yanli Su, Nathan Lanning, Margaret Gustafson, Nariyoshi Shinomiya, Ping Zhao, Brian Cao, Galia Tsarfaty, Ling-Mei Wang, Rick Hay, and George F. Vande Woude. 2005. Enhanced growth of human Met-expressing xenografts in a new strain of immunocompromised mice transgenic for human hepatocyte growth factor/scatter factor. Oncogene 24(1): 101–106. Graveel, Carrie, Yanli Su, Julie Koeman, Ling-Mei Wang, Lino Tessarollo, Michelle Fiscella, Carmen Birchmeier, Pamela Swiatek, Roderick Bronson, and George Vande Woude. 2004. Activating Met mutations produce unique tumor profiles in mice with selective duplication of the mutant allele. Proceedings of the National Academy of Sciences U.S.A. 101(49): 17198–17203. Kelloff, Gary J., Robert C. Bast, Jr., Donald S. Coffey, Anthony V. D’Amico, Robert S. Kerbel, John W. Park, Raymond W. Ruddon, Gordon J.S. Rustin, Richard L. Schilsky, Caroline C. Sigman, and George F. Vande Woude. 2004. Biomarkers, surrogate end points, and the acceleration of drug development for cancer prevention and treatment: an update. Clinical Cancer Research 10(11): 3881–3884. Lee, Chong-Chou, Andrew J. Putnam, Cindy K. Miranti, Margaret Gustafson, Ling-Mei Wang, George F. Vande Woude, and Chong-Feng Gao. 2004. Overexpression of sprouty-2 inhibits HGF/SF-mediated cell growth, invasion, migration, and cytokinesis. Oncogene 23(30): 5193–5202. Shinomiya, Nariyoshi, Chong Feng Gao, Qian Xie, Margaret Gustafson, David J. Waters, Yu-Wen Zhang, and George F. Vande Woude. 2004. RNA interference reveals that ligand-independent Met activity is required for tumor cell signaling and survival. Cancer Research 64(21): 7962–7970. Vande Woude, George F., Gary J. Kelloff, Raymond W. Ruddon, Han-Mo Koo, Caroline C. Sigman, J. Carl Barrett, Robert W. Day, Adam P. Dicker, Robert S. Kerbel, David R. Parkinson, and William J. Slichenmyer. 2004. Reanalysis of cancer drugs: old drugs, new tricks. Clinical Cancer Research 10(11): 3897–3907. Zhang, Yu-Wen, Carrie Graveel, Nariyoshi Shinomiya, and George F. Vande Woude. 2004. Met decoys: will cancer take the bait? Cancer Cell 6(1): 5–6.
From left to right, back row: Zhang, Su, Gao, DeGroot, Staal middle row: Hay, Xie, Tesfay, Kaufman, Moshkovitz front row: Reed, Vande Woude, Graveel
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Laboratory of Tumor Metastasis and Angiogenesis Craig P. Webb, Ph.D. Dr. Webb received his Ph.D. in cell biology from the University of East Anglia, England, in 1995. He then served as a postdoctoral fellow in the laboratory of George Vande Woude in the Molecular Oncology Section of the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute–Frederick Cancer Research and Development Center, Maryland (1995–1999). Dr. Webb joined VARI as a Scientific Investigator in October 1999. Laboratory Members
Staff Jennifer Bromberg-White, Ph.D. Jeremy Miller, Ph.D. David Monsma, Ph.D. Emily Eugster, M.S. Sujata Srikanth, M.Phil. Meghan Sheehan, B.S.
Visiting Scientist Gustavo Cumbo-Nacheli, M.D.
Research Interests Tumor metastasis e use model systems ranging from cellbased assays to preclinical animal Metastasis accounts for the majority of models and clinical subjects to identify cancer-related mortalities. The active recruitment and validate biomarkers and therapeutic targets of of tumor vasculature, termed angiogenesis, aggressive cancer. The ability to navigate is integral to both tumor growth and metastasis. seamlessly across these diverse model systems Our laboratory currently uses both in vitro and in (for example, comparing data from mouse vivo systems to study metastasis and angiogenesis. models to that from human clinical trials) and Using a variety of molecular technologies, we between the various molecular platforms is have identified genomic and proteomic correlates essential in optimizing the translational research of metastatic disease that may be future pipeline (Fig. 1). Our goal is to efficiently biomarkers and/or molecular targets for diagnosis translate our investigational findings into clinical practice, initially in the areas of improved diagnostics and pharmacogenomics. Our discoveries of new, potentially “drugable” targets is being coupled with siRNA technologies to verify the functional validity of targets in preclinical models. Companion diagnostics are also being developed that could be used to assess drug efficacy in patients. We are beginning to partner with pharmaceutical companies to validate new therapeutic areas for existing drugs, as well as to test predictions of optimal drug combinations. At some point in the near future, accurate diagnosis of Figure 1. Overview of translational research. Commencing with disease will naturally transition to human research subjects, we can readily transition through specimen appropriate treatment. and data collection, data analysis, preclinical models, and ultimately
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clinical trials.
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samples, we aim to identify molecular correlates of therapeutic response and disease progression.
and treatment. Using our proprietary informatics system (described below), we have identified several genes that distinguish normal from abnormal colon tissue and predict the metastatic outcome of patients with colorectal cancer. The potential diagnostic applications of these data are currently being pursued in a larger cohort of patients. Our findings to date suggest we can accurately diagnose colon cancer in pathological samples and, moreover, predict the likelihood of metastatic relapse well in advance of clinical presentation.
Systems biology XenoBase (patent pending) is a fully integrated genomic/proteomic/medical informatics system that includes analysis and annotation tools. Raw data from molecular analyses can be associated with specimens and subjects of interest, and comparative analysis can be performed on data across platforms and species. XenoBase allows for direct correlation between the subject, sample, and experimental parameters and the molecular data. Literature-based and gene ontology annotation software have been incorporated, along with specific metrics for biomarker and target discovery. Current therapeutics that specifically target molecular aberrations can also readily be identified (Fig. 2). Thus, XenoBase represents an integrated system for basic bimolecular research, clinical diagnostics, and new pharmacogenomic strategies for the future.
In addition, we are using laser capture microdissection in conjunction with genomic and proteomic technologies to identify key tumor-host interactions during metastatic progression, with emphasis on identifying the molecular factors that contribute to metastatic dormancy in the liver. A number of candidate genes are now being pursued as potential targets of future treatments. For this purpose, we have developed retroviral and lentiviral systems for delivering small hairpin RNA (shRNA) molecules that target candidate genes. We are knocking-out the expression of several potential mediators of the metastatic phenotype in human tumor cell lines and assessing the effects in orthotopic murine xenografts.
XenoBase is written primarily in C#.NET and uses a rich Windows GUI for robust client interaction. All statistical algorithms are written in C++ and optimized for a Windows 32-bit operating system. This approach combines the speed of C++ with the easy maintenance of the .NET framework. The application itself is structured into four primary layers. The GUI layer allows for client interaction with the system. The Controller layer provides the bulk of the code and is responsible for controlling interactions between the GUI, the algorithms, and the database. The Algorithm components are part of the controller layer but are segmented to work independently and are optimized for performance using C++. Finally, the Database layer houses the database I/O code and â&#x20AC;&#x153;plumbingâ&#x20AC;? necessary to keep the application database independent and allow the transactional objects to interact without the overhead of database-specific calls. The code itself uses a standard .NET exception handling framework and is documented internally using code-blocks and inline comments. The use of C#.NET allows for the automatic generation of both an object model and API documentation.
Multiple myeloma Some 40,000 Americans are living with multiple myeloma, and deaths are over 11,000 per year, usually within three years of diagnosis. Treatment of this highly aggressive cancer is extremely limited. Without in-depth study into the molecular causes of multiple myeloma, near-term advances in diagnosis and treatment are unlikely. At the end of 2002, with the generous support of the McCarty Foundation and Ralph Hauenstein, we initiated the development of a dedicated multiple myeloma research laboratory (MMRL). Our specific goal is to use our unique integrated approach to identify optimal treatments for patients. We are collaborating with Keith Stewart at the Princess Margaret Hospital, Toronto, as well as with local hematologists, oncologists, pathologists, and other specialists. Through the collection of detailed clinical information such as treatment response (efficacy and toxicity), coupled with single nucleotide polymorphism, gene expression, and proteomic analysis of collected
The system was built with expandability in mind. The layered approach allows new user interfaces (Internet application, PDA, web 54
Figure 2. Molecular networks associated with aggressive mesothelioma. This figure shows the interconnectivity between genes associated with rapidly progressing mesothelioma. Mapping genomic correlates of disease to highly curated molecular pathways can identify the underlying molecular mechanisms of the disease. This information could be used for diagnostic applications, as well as identification of key steps that may represent intervention points for treatment. Here, EGFR was identified as a key convergence point that appears hyperactivated in aggressive mesothelioma. Pathway mapping was generated using MetaCoreâ&#x201E;˘ (GeneGo, Inc., St. Joseph, MI). For more information on this tool, see http://genego.com).
variety of databases, including Microsoft SQL Server, IBM DB/2, Oracle, and MySQL. Finally, the system makes use of a database-driven storage system and is database-independent. Its design includes scripts for database creation and baseline data setup and uses ODBC (OLEDB) for rapid execution and flexible database binding.
services, etc.) to be developed without redeveloping core functionality. The .NET framework provides ready access to development tools and plug-ins, speeding development of new features and allowing easy integration with virtually any software platform. The use of ADO.NET allows for seamless integration with a
External Collaborators James R. Baker, University of Michigan Health System, Ann Arbor, Michigan Lonson L. Barr, Michigan State University College of Osteopathic Medicine, Grand Rapids, Michigan Andrej Bugrim, GeneGo, Inc., St. Joseph, Michigan Alan D. Campbell, Spectrum Health Cancer Center, Grand Rapids, Michigan Sandra L. Cottingham, Pamela G. Kidd, Susan M. Mammina, and Timothy J. Pelkey, Spectrum Health, Pathology & Laboratory Medicine, Grand Rapids, Michigan Alan T. Davis, Michigan State University and Spectrum Health, Grand Rapids, Michigan Michael Dobbs, Anthony J. Foster, Pamela Grady, Thomas J. Monroe, Linda C. Pool, Deborah RitzHolland, Marcy Ross, Angela R. Tiberio, and Michael J. Warzynski, Spectrum Health, Grand Rapids, Michigan 55
Timothy Fitzgerald, St. Mary’s Mercy Medical Center, Grand Rapids, Michigan Neal Goodwin, ProNAi, Kalamazoo, Michigan Jason Joseph, Order Streams Management, Inc., Grand Rapids, Michigan Donald G. Kim and Martin A. Luchtefeld, The Ferguson Clinic, Grand Rapids, Michigan David E. Langholz, Richard F. McNamara, and Timothy C. Vander Meulen, West Michigan Heart, Grand Rapids, Michigan Eric P. Lester, Oncology Care Associates, P.L.L.C., St. Joseph, Michigan Martin McMahon, University of California, San Francisco John B. O’Donnell, Grand Rapids Medical Education & Research Center, Grand Rapids, Michigan Gilbert S. Omenn, University of Michigan Medical School, Ann Arbor, Michigan Leon Oostendorp, Towers Surgeons, P.C., Grand Rapids, Michigan Timothy J. O’Rourke, Cancer & Hematology Centers of Western Michigan, P.C., Grand Rapids, Michigan Harvey I. Pass, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan Keith Stewart, McLaughlin Centre for Molecular Medicine, Toronto, Canada Annette Thelen, Michigan State University, East Lansing Guenter Tusch, Grand Valley State University, Allendale, Michigan Recent Publications Creighton, C.J., J.L. Bromberg-White, D. Misek, D. Monsma, F. Brichory, R. Kuick, T.J. Giordano, W. Gao, G.S. Omenn, C.P. Webb, and S.M. Hanash. In press. Reciprocal tumor-host interactions revealed by gene expression profiling of lung adenocarcinoma xenografts. Molecular Cancer Therapeutics. Bromann, Paul A., Hasan Korkaya, Craig P. Webb, Jeremy Miller, Tammy L. Calvin, and Sara A. Courtneidge. 2005. Platelet-derived growth factor stimulates Src-dependent mRNA stabilization of specific early genes in fibroblasts. Journal of Biological Chemistry 280(11): 10253–10263. Webb, Craig P., and Harvey I. Pass. 2004. Translational research: from accurate diagnosis to appropriate treatment. Journal of Translational Medicine 2: 22 pp.
From left to right: Bromberg-White, Monsma, Sheehan, Cumbo-Nacheli, Miller, Eugster, Srikanth, Webb
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High-throughput processing of antibody microarrays. Microscope slides containing 12 (A) or 48 (B) identical microarrays per slide were produced, and each microarray was incubated with a different sample. The insets show microarrays containing 288 spots (from A) and 96 spots (from B). The arrays were separated by a hydrophobic wax border that was imprinted onto the slide using a custom-built device (patent pending). The device uses an interchangable printing cartridge that can be designed to imprint any user-defined pattern onto a slide. The ability to process multiple samples on one slide increases the throughput and decreases the cost of the experiments and enables large-scale projects such as clinical biomarker studies. (Brian Haab)
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Laboratory of Chromosome Replication Michael Weinreich, Ph.D. Dr. Weinreich received his Ph.D. in biochemistry from the University of Wisconsin–Madison in 1993. He then was a postdoctoral fellow in the laboratory of Bruce Stillman, director of the Cold Spring Harbor Laboratory, New York, from 1993 to 2000. Dr. Weinreich joined VARI as a Scientific Investigator in March 2000. Laboratory Members
Staff Carrie Gabrielse, B.S. Jeffrey Kasperski, B.S.
Jessica Lanning, B.S.
Students Charles Miller, B.S. Amber Crampton
Research Interests replicative complex, or pre-RC. ORC, Cdc6p, Cdt1p, and the MCM complex are together required to form the pre-RC. Additional proteins associate with the origin during G1, and then this large complex of proteins is activated to form bidirectional replication forks by the Cdc7p-Dbf4p protein kinase. The process of origin activation is not understood.
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e are studying the initiation of chromosomal DNA replication in budding yeast and in human cells. The initiation of DNA synthesis occurs at multiple replication origins throughout the genome within S-phase, but each replication origin is activated only once per cell cycle. Initiation is precisely controlled because errors (such as initiating replication multiple times from a single origin) would cause genome amplification and instability. Since DNA replication is essential for cell division and genome instability is a property of many cancer cells, we are also investigating the aberrant regulation of initiation factors in cancer.
Does chromatin structure affect initiation? Cdc6p is a critical, limiting factor for assembly of the pre-RC. We have previously shown that Cdc6p interacts with ORC and that its essential activity requires a functional ATP-binding motif. A cdc6-4 mutant that alters the ATP binding domain is temperature sensitive (ts), and we have used this property to isolate suppressors of the cdc6-4 ts. Loss-of-function alleles within the silent information regulators (SIR2–4) suppress the cdc64 ts in varying degrees. Deletion of SIR2 gives the best suppression (Fig. 2). We have also shown that the loss of SIR2 suppresses several replication initiation mutants but that its function is originspecific. Sir2p is a histone deacetylase required for the formation of an altered (“silenced”) chromatin domain at several transcriptionally silent loci in the budding yeast. Therefore, one model to explain our finding proposes that histone acetylation stimulates pre-RC assembly. We are currently investigating the potential mechanism by which Sir2p inhibits Cdc6p function and also how the Sir2p origin specificity is achieved.
Initiation occurs at very well defined sequences in the budding yeast, so we are using this organism as a genetic model. Initiation requires at least three steps (see Fig. 1). The first is origin marking by ORC, a six-subunit complex that recognizes conserved DNA sequence elements in all origins. The second step is the assembly of a large macromolecular complex called the pre-
Figure 1. Model for the initiation of DNA replication.
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CDC7 kinase is required for DNA replication and repair Another effort in the lab is to understand how the two-subunit Cdc7p-Dbf4p kinase triggers replication initiation. We have isolated mutants of the kinase that are proficient in DNA replication but defective in DNA repair. Dbf4p is phosphorylated following inhibition of DNA replication in a MEC1- and RAD53-dependent manner and this phosphorylation appears to inhibit Cdc7p-Dbf4p kinase activity. MEC1 is a homologue of the human ATM/ATR (PI3-related) kinases that are key regulators of the response to DNA damage. RAD53 is the homologue of the human Cds1/Chk2 checkpoint kinase. Genetic data suggest that RAD53 is required for survival of our Cdc7p-Dbf4p kinase mutants and may promote an alternative function of this kinase, i.e., to aid recovery from DNA damage-induced replication arrest. Determining how the DNA damage checkpoint pathway alters the activity of Cdc7p-Dbf4p kinase or modulates DNA replication and repair is an ongoing and exciting area of research.
A.
B. WT cdc6-4 cdc6-4 orc1∆N cdc6-4 sir1∆ cdc6-4 sir2∆ cdc6-4 sir3∆ cdc6-4 sir4∆ 25˚C
37˚C
Figure 2. A) Representation of Cdc6p showing the conservation of eight motifs (II through SensII) within the AAA+ family of ATP binding proteins. A change of K114A within the “Walker A” ATP binding motif results in the cdc6-4 ts allele. B) Deletion of SIR2, SIR3, or SIR4 suppresses the cdc6-4 ts. Serial dilutions of wild-type, cdc6-4, and various cdc6-4 double-mutant strains were spotted onto plates at low and high temperature to determine growth.
From left to right, standing: Kasperski, Weinreich, Miller; seated: Crampton, Gabrielse
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Laboratory of Cell Signaling and Carcinogenesis Bart O. Williams, Ph.D. Dr. Williams received his Ph.D. in biology from Massachusetts Institute of Technology in 1996. For three years, he was a postdoctoral fellow at the National Institutes of Health in the laboratory of Harold Varmus, former Director of NIH. Dr. Williams joined VARI as a Scientific Investigator in July 1999. Laboratory Members
Staff Charlotta Lindvall-Weinreich, M.D., Ph.D. Troy A. Giambernardi, Ph.D. Katia Bruxvoort, B.S. Holli Charbonneau, B.S. Cassandra Zylstra, B.S.
Students Nicole Evans Jose Toro
Research Interests we created mice carrying an osteoblast-specific deletion of β-catenin. These mice die within five weeks of birth due to profound deficiencies in bone development. A reciprocal experiment, in which mice missing the Apc gene specifically in osteoblasts (and therefore expressing elevated levels of β-catenin), was also performed. These mutant mice again died very early after birth, and
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y laboratory is interested in understanding how alterations in the Wnt signaling pathway cause human disease. Wnt signaling is an evolutionarily conserved process that has been adapted to function in the differentiation of most tissues within the body. One of the long-term goals of our laboratory is to understand how specificity is generated for the different signaling pathways.
Recently, we have focused on understanding the role of Wnt signaling in bone formation. We are interested not only in normal bone development, but also in whether aberrant Wnt signaling plays a role in the predisposition of some common tumor types (for example prostate, breast, lung, and renal tumors) to metastasize to and grow in the bone. The long-term goal is to provide insights that could be used in developing strategies to lessen the morbidity and mortality associated with skeletal metastasis. Wnt signaling in normal bone development Mutations in the Wnt receptor, Lrp5, have been causally linked to alterations in human bone development. We have characterized a mouse strain deficient in Lrp5 that recapitulates the lowbone-density phenotype seen in human patients having this deficiency. We have further shown that mice carrying mutations in both Lrp5 and the related Lrp6 protein have even more-severe defects in bone density.
Figure 1. Bone defects in mice carrying osteoblast-specific mutations in β-catenin and/or Apc. Top set of three: micro-CT of femurs from a) a 30-day-old wild-type mouse, b) a mouse with an osteoblast-specific deletion of β-catenin or c) a mouse lacking both Apc and β-catenin in osteoblasts. Lower two: micro-CT of femurs from d) 12-day-old mice either wild type or e) carrying an osteoblast-specific deletion of Apc. Note the presence of severe osteoporosis in mice lacking β-catenin (b) and osteopetrosis in those lacking Apc in osteoblasts (e). The image in (c) demonstrates that loss of β-catenin is epistatic to the loss of Apc in osteoblasts.
We also tested whether Lrp5 deficiency causes changes in bone density due to aberrant signaling through β-catenin (Fig. 1). To do this, 60
We have created mice with a prostatespecific deletion of the Apc gene. These mice develop fully penetrant prostate hyperplasia by four months of age, and these tumors progress to frank carcinomas by seven months. We are currently assessing whether these tumors are androgen-independent, and we are evaluating whether loss of Apc can synergize with other genetic alterations to produce higher-grade prostate carcinoma. It is our ultimate goal to test whether loss of activation of β-catenin signaling can lead to a mouse model of prostate cancer with skeletal metastases.
they showed a dramatic overgrowth of bone to the point where very little marrow cavity was present. Our current work in this project is aimed at addressing the molecular mechanisms that underlie these phenotypes. We have isolated osteoblasts from these mice and are analyzing them in tissue culture to determine their abilities to produce and mineralize osteoid. Also, we are identifying potential downstream mediators of Wnt signaling in osteoblasts via microarraybased expression analysis. As a result of this work (done in collaboration with Tom Clemens), we found that alterations in Wnt/β-catenin signaling in osteoblasts led to changes in the expression of RANKL and osteoprotegerin (OPG). Consistent with this, histomorphometric evaluation of bone in the mice with osteoblastspecific deletions of either Apc or β-catenin revealed significant alterations in osteoclastogenesis. We are currently working to address how other genetic alterations linked to Wnt/βcatenin signaling affect bone development and osteoblast function.
We are also examining the roles of Lrp5 and Lrp6 in normal mammary development. We have found that mice lacking these genes have delays in normal mammary development. We are currently assessing the temporal and spatial organization of Lrp6 and Lrp5 expression during this process. We are also addressing the relative roles of Lrp6 and Lrp5 in Wnt1-induced mammary carcinogenesis. We have found that deficiency in Lrp5 dramatically inhibits the development of mammary tumors in this context. The tumors that do develop have altered morphology. We are testing the hypothesis that this inhibition of tumorigenesis is associated with changes in mammary stem cells.
Wnt signaling in cancer Activation of the Wnt signaling pathway occurs in a significant percentage of prostate carcinomas. In some cases this is associated with activating mutations in the β-catenin genes, while in others there is a loss of APC. Two hallmarks of advanced prostate cancer are skeletal osteoblastic metastasis and the androgenindependent survival of tumor cells. The association of Wnt signaling with bone growth, plus the fact that β-catenin can bind to the androgen receptor and make it more susceptible to activation with steroid hormones other than dihydrotestosterone, make Wnt signaling an attractive candidate to explain some phenotypes associated with advanced prostate cancer.
VARI mutant mouse repository With partial support from the Michigan Animal Models Consortium, my laboratory maintains a repository of mutant mouse strains to support the general development of animal models of human disease. The repository includes strains that can be used with the RCAS/TVA retroviral gene targeting system, which my laboratory has used to develop more efficient ways of accurately and efficiently modeling human diseases in mice.
External Collaborators Caroline Alexander, University of Wisconsin–Madison Mary Bouxsein, Beth Israel Deaconess Medical Center, Boston, Massachusetts Thomas Clemens, University of Alabama–Birmingham Marie-Claude Faugere, University of Kentucky, Lexington Peter Igarashi, University of Texas–Southwestern Medical School, Dallas Michael T. Lewis and Yi Li, Baylor Breast Center, Houston, Texas Matthew Warman, Case Western Reserve University, Cleveland, Ohio 61
Recent Publications Ai, Minrong, Sheri L. Holmen, Wim van Hul, Bart O. Williams, and Matthew W. Warman. 2005. Reduced affinity to and inhibition by DKK1 form a common mechanism by which high bone mass–associated missense mutations in LRP5 affect canonical Wnt signaling. Molecular and Cellular Biology 25(12): 4946–4955. Holmen, Sheri L., Scott A. Robertson, Cassandra R. Zylstra, and Bart O. Williams. 2005. Wnt-independent activation of ß-catenin mediated by a Dkk-1-Frizzled 5 fusion protein. Biochemical and Biophysical Research Communications 328(2): 533–539. Holmen, Sheri L., Cassandra R. Zylstra, Aditi Mukherjee, Robert Sigler, Marie-Claude Faugere, Mary Bouxsein, Lianfu Deng, Thomas Clemens, and Bart O. Williams. 2005. Essential role of ß-catenin in postnatal bone acquisition. Journal of Biological Chemistry 280(22): 21162–21168. Qian, Chao-Nan, Jared Knol, Peter Igarashi, Fangmin Lin, Uko Zylstra, Bin Tean Teh, and Bart O. Williams. 2005. Cystic renal neoplasia following conditional inactivation of Apc in mouse renal tubular epithelium. Journal of Biological Chemistry 280(5): 3938–3945. Robertson, Scott A., Jacqueline Schoumans, Brendan D. Looyenga, Jason A. Yuhas, Cassandra R. Zylstra, Julie M. Koeman, Pamela J. Swiatek, Bin T. Teh, and Bart O. Williams. 2005. Spectral karyotyping of sarcomas and fibroblasts derived from Ink4a/Arf-deficient mice reveals chromosomal instability in vitro. International Journal of Oncology 26(3): 629–634. Holmen, Sheri L., Troy A. Giambernardi, Cassandra R. Zylstra, Bree D. Buckner-Berghuis, James H. Resau, J. Fred Hess, Vaida Glatt, Mary L. Bouxsein, Minrong Ai, Matthew L. Warman, and Bart O. Williams. 2004. Decreased BMD and limb deformities in mice carrying mutations in both Lrp5 and Lrp6. Journal of Bone and Mineral Research 19(12): 2033–2040. Spike, Benjamin T., Alexandra Dirlam, Benjamin C. Dibling, James Marvin, Bart O. Williams, Tyler Jacks, and Kay F. Macleod. 2004. The Rb tumor suppressor is required for stress erythropoiesis. EMBO Journal 23(21): 4319–4329.
From left to right: Williams, Charbonneau, Toro, Zylstra, Giambernardi, Bruxvoort
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Laboratory of Structural Sciences H. Eric Xu, Ph.D. Dr. Xu went to Duke University and the University of Texas Southwestern Medical Center, where he earned his Ph.D. in molecular biology and biochemistry. Following a postdoctoral fellowship with Carl Pabo at MIT, he moved to GlaxoWellcome in 1996 as a research investigator of nuclear receptor drug discovery. Dr. Xu joined VARI as a Senior Scientific Investigator in July 2002. Laboratory Members
Staff Schoen Kruse, Ph.D. Yong Li, Ph.D. David Tolbert, Ph.D. X. Edward Zhou, Ph.D.
Jennifer Daughtery, B.S. Amanda Kovach, B.S. Kelly Suino, B.S. Tricia Velting, B.S.
Visiting Scientist Ross Reynolds, Ph.D.
Research Interests acids, the lipid-lowering fibrate drugs, and the new anti-diabetic drugs called glitazones. We have also determined crystal structures of these receptors bound to co-activators or co-repressors. These structures have provided a framework for understanding the mechanisms of agonists and antagonists, as well as the mechanisms for recruitment of co-activators and co-repressors by nuclear receptors. We are now developing this project beyond the structures of the ligandbinding domains and into defining the structures of large PPAR fragment/DNA complexes.
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ur laboratory is using x-ray crystallography and molecular biology to study structures and functions of key protein complexes that are important in basic biology and in drug discovery relevant to human diseases such as cancers and diabetes. Currently we are focusing on nuclear hormone receptors and the Met tyrosine kinase receptor. The nuclear hormone receptors Nuclear hormone receptors form a large family of ligand-regulated and DNA-binding transcriptional factors, which include receptors for classic steroid hormones (such as estrogen, progesterone, androgens, and glucocorticoids), as well as receptors for peroxisome proliferator activators, vitamin D, vitamin A, and thyroid hormones. These classic receptors are among the most successful targets in the history of drug discovery. Almost every one has one or more synthetic ligands currently being used as medicines. In the last two years, we have developed the following projects centering on the structural biology of nuclear receptors.
The human glucocorticoid receptor The human glucocorticoid receptor (GR) is a key regulator of energy metabolism and of homeostasis of the immune system. The GR is also a classic target of drug discovery due to its association with numerous pathological conditions. There are more than 10 GR ligands (including dexamethasone) that are currently used for treating such diverse medical conditions as asthma, allergy, autoimmune diseases, and cancer. At the molecular level, GR can function either as a transcriptional activator or repressor. Both functions are tightly regulated by small ligands that bind to the GR LBD. To explore the molecular mechanisms of GR ligand binding and signaling, we determined a crystal structure of the GR LBD bound to dexamethasone and a coactivator motif from TIF2. The structure revealed a novel LBD-LBD dimer interface, an unexpected charge clamp responsible for sequence-specific binding of co-activators, and a unique ligand-binding pocket that accounts for
Peroxisome proliferator–activated receptors The peroxisome proliferator–activated receptors (PPARα, δ, and γ) are the key regulators of glucose and fatty acid homeostasis and as such are important therapeutic targets for cardiovascular disease, diabetes, and cancer. To understand the molecular basis of ligandmediated signaling, we have determined crystal structures of each PPAR’s ligand-binding domain (LBD) bound to diverse ligands including fatty 63
the specific recognition of diverse GR ligands. Currently we are studying receptor-ligand interactions by crystallizing GR with various steroid or nonsteroid molecules. The human androgen receptor The androgen receptor (AR) is the central molecule in the development and progression of prostate cancer, and as such it serves as the molecular target of anti-androgen therapy. However, the majority of prostate cancer patients develop resistance to anti-androgen therapy, mostly due to mutations in this hormone receptor that alter the three-dimensional structure of the receptor. These hormone-independent cells are highly aggressive and are responsible for most deaths from prostate cancer. In this project, we are aiming to determine the structures of the mutated AR proteins that alter the response to anti-hormone therapy. In collaboration with Donald MacDonnell, we are working on the crystal structure of the full-length AR/DNA complex.
Figure 1. Structural comparison of CAR vs. PXR. The ligand binding pockets are shown as the pink surface. The AF-2 helix of the receptors is in red and the co-activator helix is in purple.
abolish SF-1/co-activator interactions and reduce SF-1 transcriptional activity. These findings establish that SF-1 is a ligand-dependent receptor and suggest an unexpected link between nuclear receptors and phospholipid signaling pathways. We can expect more surprises as structural work continues on the remaining orphan receptors. The Met tyrosine kinase receptor
Structural genomics of nuclear receptor ligand binding domains
Met is a tyrosine kinase receptor that is activated by hepatocyte growth factor/scatter factor (HGF/SF). Aberrant activation of the Met receptor has been linked with the development and metastasis of many types of solid tumors and has been correlated with poor clinical prognosis. HGF/SF has a modular structure with an Nterminal domain, four kringle domains, and an inactive serine protease domain. The structure of the N-terminal domain with a single kringle domain (NK1) has been determined; less is known about the structure of the Met
The LBD of nuclear receptors contains key structural elements that mediate the receptors’ ligand-dependent regulation, and as such, the LBD has been the focus of intense structural studies. There are only a few nuclear receptors for which the LBD structure remains unsolved. In the past two years, we have focused on structural characterization of two of these “orphan” receptors: constitutive androstane receptor (CAR) and steroidogenic factor-1 (SF-1). The CAR structure reveals a compact LBD fold that contains a small pocket only half the size of the pocket in PXR, a closely related receptor (Fig. 1). The constitutive activity of CAR appears to be mediated by a novel linker helix between the Cterminal AF-2 helix and helix 10. On the other hand, SF-1 is regarded as a ligand-independent receptor, but its LBD structure reveals the presence of a phospholipid ligand in a surprisingly large pocket, more than twice the size of the pocket in the mouse LRH-1, a closely related receptor (Fig. 2). The bound phospholipid is readily exchanged and modulates SF-1 interactions with co-activators. Mutations designed to reduce the size of the SF-1 pocket or to disrupt hydrogen bonds with the phospholipid
Figure 2. Ribbon representation of the SF-1/ phospholipid/SHP complex in two views separated by 90˚. SF-1 is in red and the SHP ID1 motif is in yellow. The bound phospholipid ligand is shown in a space-filling representation, with carbon, oxygen, nitrogen, and phosphate depicted as green, red, blue, and purple, respectively.
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extracellular domain. The molecular basis of the Met-HGF/SF interaction and the activation of Met signaling by this interaction remain poorly understood. In collaboration with George Vande
Woude and Ermanno Gherardi, we are developing this project to solve the crystal structure of the Met receptor/HGF complex.
External Collaborators Doug Engel, University of Michigan, Ann Arbor Ermanno Gherardi, University of Cambridge, U.K. Steve Kliewer, University of Texas Southwestern Medical Center, Dallas Millard Lambert, GlaxoSmithKline Inc., Research Triangle Park, North Carolina Donald MacDonnell, Duke University, Durham, North Carolina Stoney Simmons, National Institutes of Health, Bethesda, Maryland Scott Thacher, Orphagen Pharmaceuticals, San Diego, California Brad Thompson, University of Texas Medical Branch at Galveston Ming-Jer Tsai, Baylor College of Medicine, Houston, Texas Recent Publications Li, Y., M. Choi, K. Suino, A. Kovach, J. Daugherty, S.A. Kliewer, and H.E. Xu. In press. Structural and biochemical basis for selective repression of the orphan nuclear receptor LRH-1 by SHP. Proceedings of the National Academy of Sciences U.S.A. Li, Yong, Mihwa Choi, Greg Cavey, Jennifer Daugherty, Kelly Suino, Amanda Kovach, Nathan C. Bingham, Steven A. Kliewer, and H. Eric Xu. 2005. Crystallographic identification and functional characterization of phospholipids as ligands for the orphan nuclear receptor steroidogenic factor-1. Molecular Cell 17(4): 491–502. Haffner, Curt D., James M. Lenhard, Aaron B. Miller, Darryl L. McDougals, Kate Dwornik, Olivia R. Ittoop, Robert T. Gampe, Jr., H. Eric Xu, Steve Blanchard, Valerie G. Montana, Tom G. Consler, Randy K. Bledsoe, Andrea Ayscue, and Dallas Croom. 2004. Structure-based design of potent retinoid X receptor α agonists. Journal of Medicinal Chemistry 47(8): 2010–2029. Suino, Kelly, Li Peng, Ross Reynolds, Yong Li, Ji-Young Cha, Joyce J. Repa, Steven A. Kliewer, and H. Eric Xu. 2004. The nuclear xenobiotic receptor CAR: structural determinants of constitutive activation and heterodimerization. Molecular Cell 16(6): 893–906.
From left to right: Li, Zhou, Tolbert, Daugherty, Xu, Velting, Kruse, Suino, Kovach
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Laboratory of Mammalian Developmental Genetics Nian Zhang, Ph.D. Dr. Zhang received his M.S. in entomology from Southwest Agricultural University, People’s Republic of China, in 1985 and his Ph.D. in molecular biology from the University of Edinburgh, Scotland, in 1992. From 1992 to 1996, he was a postdoctoral fellow at the Roche Institute of Molecular Biology. He next served as a postdoctoral fellow (1996) and a Research Associate (1997–1999) in the laboratory of Tom Gridley in mammalian developmental genetics at the Jackson Laboratory, Bar Harbor, Maine. Dr. Zhang joined VARI as a Scientific Investigator in December 1999. Laboratory Members
Staff Wei Ma, Ph.D. Lisheng Zhang, Ph.D. Kate Groh, B.S. Liang Kang
Student William Bond
Research Interests interacts with Hes7 to turn down Hes7 and Lfng. When the level of HES7 is down, it relieves its repression on Lfng and its own promoter, and thus the next cycle begins. We have also demonstrated that the 3′-untranslated region (UTR) is important for the rapid degradation of Lfng mRNA, which ensures accurate oscillation.
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e are interested in understanding the cellular and molecular mechanisms underlying pattern formation during embryonic development. We previously cloned and targeted the mouse Lunatic fringe (Lfng) gene, which plays an important role in embryo segmentation. Mice homozygous for the Lfng mutation suffer from severe malformation of their axial skeleton as a result of irregular somite formation during embryonic development. Lfng encodes a secreted signaling molecule essential for regulating the Notch signaling pathway in mice. We showed that Lfng expression was in response to a biological clock that oscillated once during the formation of each segment, and the failure of the Lfng mutants in responding to this clock resulted in the abnormal segmentation phenotype. We want to understand how the rhythmic expression of Lfng is controlled. Our recent studies indicate that the cyclic expression of Lfng is controlled by a negative feedback loop. The signals transmitted through the loop are mediated by the components in the Notch signaling pathway. We found that Hes7 can directly bind to the N-boxes in the 5′regulatory region of the Lfng gene and its own promoter and repress their transcription in vitro. Hes7 can also override the Notch-mediated activation of the Lfng promoter. However, our results suggest that Hes7 needs a co-factor to achieve this suppression in vivo. Our data suggests that when NOTCH1 is modified by LFNG, it becomes receptive to the signal from DLL3, thus activating its downstream target. This target
Germ cell development The second focus of our laboratory is on germ cell development, particularly the mechanisms that govern germ cell migration, survival, spermatogonial stem cell renewal, and differentiation, as well as their implications for human disease. It is unclear how spermatogonial stem cells are regenerated during the entire reproductive life in mammals. Previous studies on the nematode Caenorhabditis elegans have shown that the Notch/lin12-mediated signal transduction pathway is important if germ cells are to remain in an undifferentiated state. Mutations that compromise this pathway force germ cells to enter meiosis earlier than normal. A constitutively activated signal prevents germ cells from entering meiosis, resulting in overproliferation of germ cells, a phenotype called “germ cell tumor.” Given the fact that some members in the Notch signaling pathway are expressed in the testis, we speculate that Notch signaling may play a similar role in spermatogonial differentiation in mammals. We will further examine the role Notch signaling may play during spermatogenesis using transgenic animals and conditional gene targeting. 66
the wild type. We have mapped the atrichosis mutation to a 270-kb region on chromosome 10. We are now screening the candidate genes by transgenic rescue and direct sequencing.
We are also studying spontaneous mutations that cause sterility. In mice, primordial germ cells (PGCs) are differentiated from the epiblasts during an early embryonic stage. After their formation, they migrate through the dorsal mesentery and enter the genital ridge, where they collaborate with the somatic gonad cells to form the gonads. The PGCs proliferate during their migration. We are studying the spontaneous mutation atrichosis (at), which causes male and female sterility. Preliminary data suggest that this mutation affects fetal germ cell proliferation. We found that by 12.5 dpc, there already are significantly fewer germ cells in the gonads of atrichosis embryos relative to
Another mutation we are studying is in the sks gene; this mutation affects normal meiosis in both sexes. Our results indicate that sks is required for the metaphase/anaphase transition in meiosis I. In the sks mutant, homologous chromosomes fail to separate; therefore, meiosis is stopped at MI. We have demonstrated that this failure is due to the inability of the sks mutant to degrade securin in the primary spermatocytes and possibly in the oocytes.
From left to right, back row: Kang, Ma, L. Zhang, Groh; front row: N. Zhang, Bond
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Double staining of protein and RNA in mouse testis This is a section of mouse testis stained with anti-MPM-2 antibody (green) and an RNA probe (red) to label the spermatocytes. This image is from studies of germ cell development and the implications for human disease. (Nian Zhang)
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Daniel Nathans Memorial Award
Daniel Nathans Memorial Award The Daniel Nathans Memorial Award was established in memory of Dr. Daniel Nathans, a distinguished member of our scientific community and a founding member of VARI’s Board of Scientific Advisors. We established this award to recognize individuals who emulate Dan and his contributions to biomedical and cancer research. It is our way of thanking and honoring him for his help and guidance in bringing Jay and Betty Van Andel’s dream to reality. The Daniel Nathans Memorial Award was announced at our inaugural symposium, “Cancer & Molecular Genetics in the Twenty-First Century,” in September 2000.
2004 Award to Dr. Brian Druker The 2004 recipient of the Daniel Nathans Memorial Award is Brian Druker, M.D., of the Oregon Health & Science University Cancer Institute and the Howard Hughes Medical Institute. Dr. Druker’s research focuses on translating knowledge of the molecular pathogenesis of cancer into specific therapies, and he is investigating the optimal use of molecularly targeted agents. Dr. Druker will visit Grand Rapids in September 2005 to deliver two lectures, one directed to a scientific audience and the second directed to the general public.
Previous Award Recipients 2000 2001 2002 2003
Richard D. Klausner, M.D. Francis S. Collins, M.D., Ph.D. Lawrence H. Einhorn, M.D. Robert A. Weinberg, Ph.D.
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Postdoctoral Fellowship Program
Postdoctoral Fellowship Program The Van Andel Research Institute provides postdoctoral training opportunities to Ph.D. scientists beginning their research careers. The fellowships help promising scientists advance their knowledge and research experience while at the same time supporting the research endeavors of VARI. The fellowships are funded in three ways: 1) by the laboratories to which the fellow is assigned; 2) by the VARI Office of the Director; or 3) by outside agencies. Each fellow is assigned to a scientific investigator who oversees the progress and direction of research. Fellows who worked in VARI laboratories in 2004 and early 2005 are listed below.
Eduardo Azucena Wayne State University, Detroit, Michigan VARI mentor: Sara Courtneidge
Chongfeng Gao Tokyo Medical and Dental University, Japan VARI mentor: George Vande Woude
Paul Bromann Northwestern University, Evanston, Illinois VARI mentor: Sara Courtneidge
Troy Giambernardi University of Texas Health Science Center, San Antonio VARI mentor: Bart Williams
Jennifer Bromberg-White Pennsylvania State University College of Medicine, Hershey VARI mentor: Craig Webb
Carrie Graveel University of Wisconsin â&#x20AC;&#x201C; Madison VARI mentor: George Vande Woude
Jun Chen West China University of Medical Sciences, Chengdu, China VARI mentor: Nian Zhang
Holly Holman University of Glasgow, U.K. VARI mentor: Arthur Alberts
Yunju Chen University of Glasgow, U.K. VARI mentor: Arthur Alberts
Hasan Korkaya International Center for Genetic Engineering and Biotechnology, New Delhi, India VARI mentor: Sara Courtneidge
Philippe Depeille University of Montpellier, France VARI mentor: Nicholas Duesbery
Schoen Kruse University of Colorado, Boulder VARI mentor: Eric Xu
Mathew Edick University of Tennessee, Memphis VARI mentor: Cindy Miranti
Xudong Liang Qinghai Medical University, Xining, China VARI mentor: Nicholas Duesbery
Kathryn Eisenmann University of Minnesota, Minneapolis VARI mentor: Arthur Alberts
Phumzile Loudidi Cambridge University, England VARI mentor: Eric Xu
Kunihiko Futami Tokyo University of Fisheries, Japan VARI mentor: Bin Teh
Wei Ma Chinese Academy of Science, Beijing VARI mentor: Nian Zhang
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Donald Pappas, Jr. Louisiana State University, Baton Rouge VARI mentor: Michael Weinreich
Min-Han Tan National University of Singapore, Singapore VARI mentor: Bin Teh
Ian Pass University of Dundee, Scotland VARI mentor: Sara Courtneidge
Rebecca Uzarski Michigan State University, East Lansing VARI mentor: Sara Courtneidge
Michael Shafer Michigan State University, East Lansing VARI mentor: Brian Haab
Pengfei Wang Fourth Military Medical University, Xian, China VARI mentor: Bin Teh
Muthu Shanmugam National University of Singapore, Singapore VARI mentor: Brian Haab
Qian Xie Fudan University, Shanghai, China VARI mentor: George Vande Woude
Paul Spilotro St. George University, Grenada VARI mentor: Nicholas Duesbery
Chun Zhang Tokyo Medical and Dental University, Japan VARI mentor: Bin Teh
Suganthi Sridhar Southern Illinois University, Carbondale VARI mentor: Cindy Miranti
Lisheng Zhang Chinese Academy of Science, Beijing VARI mentor: Nian Zhang
Jun Sugimura University of Morioka Medical School, Japan VARI mentor: Bin Teh
Xiaoyin Zhou University of Alabama, Birmingham VARI mentor: Eric Xu
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Student Programs
Grand Rapids Area Pre-College Engineering Program The Grand Rapids Pre-College Engineering Program (GRAPCEP) is administered by Davenport College and jointly sponsored and funded by Pfizer, Inc., and VARI. The program is designed to provide selected high school students, who have plans to major in science or genetic engineering in college, the opportunity to work in a research laboratory. In addition to training in research methods, the students also learn workplace success skills such as teamwork and leadership. The three 2004 GRAPCEP students were Lynda Gladding (Resau/Duesbery) Union High School
Ricky Gonzales (Resau/Duesbery) Ottawa Hills High School
Mehreteab Mengsteab (Xu) Creston High School
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Summer Student Internship Program The VARI student internships were established to provide college students with an opportunity to work with professional researchers in their fields of interest, to use state-of-the-art equipment and technologies, and to learn valuable people and presentation skills. At the completion of the 10-week program, the students summarize their projects in an oral presentation. From May 2004 to March 2005, VARI hosted 44 students from 17 colleges and universities in formal summer internships under the Frederik and Lena Meijer Student Internship Program and in other student positions during the year. An asterisk (*) indicates a Meijer student intern.
Aquinas College, Grand Rapids, Michigan Brent Goslin* (Weinreich)
Hope College, Holland, Michigan Marie Graves (Cavey) Wendy Johnson (Cavey) Tom LaRoche (Haab) Richard Schildhouse (Haab) Mary VerHeulen (Vande Woude)
Calvin College, Grand Rapids, Michigan Arianne Folkema* (Williams) Jason Koning (Williams) Cornell University, Ithaca, New York Susan Kloet (Teh)
Michigan State University, East Lansing Aaron DeWard (Weinreich/Alberts) Stephanie Ellison* (Vande Woude) Mia Hemmes* (Duesbery) Jennifer Kaufman (Resau) Yaojian Liu (Alberts) Charles Miller (Weinreich)
Dalhousie University, Nova Scotia Jasmine Belanger* (Haab) Grand Rapids Community College, Michigan Jose Toro (Williams)
Michigan Technological University, Houghton Hien Dang (Resau)
Grand Valley State University, Allendale, Michigan Timothy Bearup* (Cao) Jack DeGroot (Vande Woude) Nicole Evans (Williams) Erik Freiter (Miranti) Lisa Orcasitas (Duesbery) Benjamin Staal* (Vande Woude) Neil Swanson (Duesbery) Kelli VanDussen (Weinreich) Tricia Velting* (Xu)
Nanjing Medical University, China Yong-jun Jiao (Cao) Jin Zhu (Cao) Xin Wang (Cao) University of Bath, United Kingdom William Bond (Zhang) Katharine Collins (Alberts) Amber Crampton (Weinreich) Victoria Hammond (Weinreich) Amy Percival (Resau)
Grove City College, Pennsylvania Sarah Feenstra (Vande Woude)
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University of Detroit Mercy Brandon Leeser (Resau)
University of Richmond, Virginia Jeremiah NcNamara* (Webb)
University of Illinois at Urbana-Champaign Huang Tran (Resau)
Western Michigan University, Kalamazoo Kenneth Olinger* (Resau) Nicole Repair* (Miranti)
University of Michigan, Ann Arbor Benjamin Briggs* (Furge) Michael Wells* (Teh) Natalie Wolters* (Courtneidge)
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Han-Mo Koo Memorial Seminar Series
Han-Mo Koo Memorial Seminar Series This seminar series is dedicated to the memory of Dr. Han-Mo Koo, who was a VARI Scientific Investigator from 1999 until his passing in May of 2004.
2004 January Karen Vousden, Beatson Institute for Cancer Research, Glasgow, Scotland “The p53 pathway as a therapeutic target” Patrick Brophy, University of Michigan, Ann Arbor “Ureteric budding: controlling two ends of the event” February Jeffrey Settleman, Harvard Medical School, Boston, Massachusetts “Rho GTPase signaling in development” Josef Prchal, Baylor College of Medicine, Houston, Texas “Tumors, hypoxia, and polycythemic disorders” March Robert Weinberg, Whitehead Institute for Biomedical Research, Cambridge, Massachusetts The Daniel Nathans Lecture: “Mechanisms of human tumor formation” “How cancer begins” (lay audience) Mike Caliguiri, Ohio State University, Columbus “Natural killer cells: biology and clinical implications” James Basilion, Massachusetts General Hospital, Charlestown, Massachusetts “Noninvasive imaging of gene expression: imaging multiple targets simultaneously” David Frank, Harvard University, Cambridge, Massachusetts “STAT signal transduction in the pathogenesis and treatment of cancer” April Ernst-Robert Lengyel, University of Chicago, Illinois “Regulation of proteolysis by adhesion receptors” Kathleen Siminovitch, Mount Sinai Hospital, Toronto, Canada “The Wiskott-Aldrich syndrome protein: forging the link between actin and T cell activation” Jose Cibelli, Michigan State University, East Lansing “Embryonic stem cells by parthenogenesis in mammals”
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May Francesco Marincola, National Institutes of Health, Bethesda, Maryland “Anti-cancer vaccines: from bench to bedside and from bedside to bench” Robert Gallo, University of Maryland, Baltimore “HIV in the third decade — some lessons from past experiences and future prospects” June Cynthia Wetmore, Mayo Clinic, Rochester, Minnesota “Implications of Ptc haploinsufficiency for the proliferation and cell fate of neuronal precursors” July Arnold Glazier, Drug Innovation and Design, Newton, Massachusetts “Pattern recognition tumor targeting” Renata Pasqualini, M.D. Anderson Cancer Center, Houston, Texas “Translating function protein interactions into targeted therapies for cancer and obesity” September Douglas Hanahan, University of California, San Francisco “Mechanisms of angiogenesis, and anti-angiogenic therapies in mouse models of cancer” Henry Higgs, Dartmouth Medical School, Hanover, New Hampshire “Comparative molecular physiology of mammalian formin proteins: potent actin assembly factors” October Janice P. Dutcher, Our Lady of Mercy Medical Center, New York “Clinical features and treatment of metastatic renal cell cancer” Robert L. Heinrikson, Proteos, Kalamazoo, Michigan “Innovation in design and production of proteins and peptides — the Proteos approach” Qing-Xiang Sang, Florida State University, Tallahassee, Florida “Metalloproteases and nonprotease factors in early stages of tumor invasion: new hypotheses on the mutated stem cells and cancer drug resistance” November John Carpten, Translational Genomics Research Institute, Phoenix, Arizona “Searching for prostate cancer in tumor suppressor genes” December Marcos Dantus, Michigan State University, East Lansing “The development of future biomedical applications using coherent laser control” Roland S. Annan, GlaxoSmithKline, King of Prussia, Pennsylvania “A qualitative and quantitative view of phosphorylation-dependent biological function”
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2005 January Ali Shilatifard, St. Louis University, Missouri “A COMPASS and a GPS in defining molecular machinery in histone modifications, transcriptional regulation, and human cancer: the coordinates of the genome” February Hsueh-Chia Chang, University of Notre Dame, Indiana “Microfluidic technologies for cancer detection and drug delivery” Paul A. Krieg, University of Arizona, Tucson “Growth factor regulation of vascular development” March Max Wicha, University of Michigan, Ann Arbor “Stem cells in normal human breast development and cancer” Judah Folkman, Harvard Medical School, Boston, Massachusetts “Platelet angiogenic profile as an early biomarker for cancer” Robert J. Amato, The Methodist Hospital, Houston, Texas “Therapeutic updates for the management of patients with renal cell carcinoma” Aaron M. Zorn, Cincinnati Children’s Hospital Research Foundation, Ohio “Sox17 and β-catenin signaling in development” April Martin E. Hemler, Harvard Medical School and Dana-Farber Cancer Institute, Boston, Massachusetts “Cell surface molecular networking: the role of tetraspanin-enriched microdomains” Alan Mackay-Sim, Griffith University, Australia “Adult stem cells from olfactory mucosa” Ravi Salgia, University of Chicago, Illinois “Role of c-Met in lung cancer”
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Organization
Van Andel Research Institute Boards
VARI Board of Trustees David L. Van Andel, Chairman and CEO Christian Helmus, M.D. Fritz M. Rottman, Ph.D. James B. Wyngaarden, M.D.
David L. Van Andel Board of Scientific Advisors The Board of Scientific Advisors advises the CEO and the Board of Trustees, providing recommendations and suggestions regarding the overall goals and scientific direction of VARI. The members are Michael S. Brown, M.D., Chairman Richard Axel, M.D. Joseph L. Goldstein, M.D. Tony Hunter, Ph.D. Phillip A. Sharp, Ph.D.
Scientific Advisory Board The Scientific Advisory Board advises the VARI Director, providing recommendations and suggestions specific to the ongoing research, especially in the areas of cancer, genomics, and genetics. It also coordinates and oversees the scientific review process for the Instituteâ&#x20AC;&#x2122;s research programs. The members are Alan Bernstein, Ph.D. Malcolm Brenner, M.D., Ph.D. Patrick O. Brown, M.D., Ph.D. Joan Brugge, Ph.D. Webster Cavenee, Ph.D. Frank McCormick, Ph.D. Davor Solter, M.D., Ph.D. Bruce Stillman, Ph.D.
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Van Andel Research Institute Office of the Director Director
George Vande Woude, Ph.D.
Deputy Director for Clinical Programs
Deputy Director for Special Programs
Rick Hay, M.D., Ph.D.
James H. Resau, Ph.D.
Deputy Director for Research Operations
Director for Research Administration
Bin T. Teh, M.D., Ph.D.
Roberta Jones
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Van Andel Research Institute Administrator to the Director
Science Editor
Michelle Reed
David E. Nadziejka
Administration Group
From left to right, back row: Dingman, Holman, Stougaard, Ferrell, Carrigan; front row: Antio, Koo, McGrail, Novakowski
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Van Andel Institute Administrative Organization The organizational units listed below provide administrative support to both the Van Andel Research Institute and the Van Andel Education Institute. Executive Steven R. Heacock, Chief Administrative Officer and General Counsel R. Jack Frick, Chief Financial Officer Ann Schoen, Executive Assistant
Finance Timothy Myers, Controller Heather Ly, Supervisor Richard Herrick Keri Jackson Angela Lawrence Susan Raymond Kevin Tefelsky Jamie VanPortfleet
Communications and Development Patrick Kelly, Vice President John Van Fossen Dianna Davidson Andrea Nielsen Margo Pratt
Purchasing Richard Disbrow, Manager Chris Kutchinski Amy Poplaski John Waldon
Information Technology Bryon Campbell, Ph.D., Chief Information Officer David Drolett, Manager Michael Roe, Manager Tom Barney Phil Bott Kenneth Hoekman Kimberlee Jeffries Theo Pretorius Russell Vander Mey Candy Wilkerson
Facilities Samuel Pinto, Manager Jason Dawes Richard Sal Richard Ulrich Security Kevin Denhof, CPP, Chief Christen Dingman Sandra Folino Emily Young
Human Resources Linda Zarzecki, Director Margie Hoving Pamela Murray Angela Plutschouw
Glass Washing/ Media Preparation Heather Frazee Marlene Sal
Grants and Contracts Carolyn W. Witt, Director Rob Junge Sara Oâ&#x20AC;&#x2122;Neal David Ross
Contract Support Valeria Long, Librarian (Grand Valley State University) Jim Kidder, Safety Manager (Michigan State University) Raymond Rupp Patty Sund Al Troupe
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Van Andel Institute
Van Andel Research Institute
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Recent VARI Photos
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Back cover photo: Spectral karyotyping of a tumor cell line Fluorescence microscopy for spectral karyotyping (SKY) analysis of a human tumor cell line. The red light helps reduce fading in the fluorescent slides, which are light sensitive. (Julie Koeman)
The Van Andel Institute and/or its affiliated organizations (VARI and VAEI), through its responsible managers, recruits, hires, upgrades, trains, and promotes in all job titles without regard to race, color, religion, sex, national origin, age, height, weight, marital status, disability, pregnancy, or veteran status, except where an accommodation is unavailable and/or it is a bone fide occupational qualification.
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